Truncated St6galnaci Polypeptides and Nucleic Acids

ABSTRACT

The present invention features compositions and methods related to truncated mutants of ST6GalNAcI. In particular, the invention features truncated human, mouse, and chicken ST6GalNAcI polypeptides. The invention also features nucleic acids encoding such truncated polypeptides, as well as vectors, host cells, expression systems, and methods of expressing and using such polypeptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/576,433, filed Jun. 3, 2004 and U.S. Provisional Application No.60/650,011, filed Feb. 4, 2005; both of which are herein incorporated byreference for all purposes.

FIELD OF INVENTION

The present invention features compositions and methods related totruncated mutants of ST6GalNAcI. In particular, the invention featurestruncated human, mouse, and chicken ST6GalNAcI polypeptides. Theinvention also features nucleic acids encoding such truncatedpolypeptides, as well as vectors, host cells, expression systems, andmethods of expressing and using such polypeptides.

BACKGROUND OF THE INVENTION

A great diversity of oligosaccharide structures and many types ofglycopeptides are found in nature, and these are synthesized, in part,by a large number of glycosyltransferases. Glycosyltransferases catalyzethe synthesis of glycolipids, glycopeptides, and polysaccharides, bytransferring an activated mono- or oligosaccharide residue to anexisting acceptor molecule for the initiation or elongation of thecarbohydrate chain. A catalytic reaction is believed to involve therecognition of both the donor and acceptor by suitable domains, as wellas the catalytic site of the enzyme.

Many peptide therapeutics, and many potential peptide therapeutics, areglycosylated peptides. The production of a recombinant glycopeptide, asopposed to a recombinant non-glycosylated peptide, requires that arecombinantly-produced peptide is subjected to additional processingsteps, either within the cell or after the peptide is produced by thecell, where the processing steps are performed in vitro. The peptide canbe treated enzymatically to introduce one or more glycosyl groups ontothe peptide, using a glycosyltransferase. Specifically, theglycosyltransferase covalently attaches the glycosyl group or groups tothe peptide.

The extra in vitro steps of peptide processing to produce a glycopeptidecan be time consuming and costly. This is due, in part, to the burdenand cost of producing recombinant glycosyltransferases for the in vitroglycosylation of peptides and glycopeptides to produce glycopeptidetherapeutics. As the demand and usefulness of recombinantglycotherapeutics increases, new methods are required in order to moreefficiently prepare glycopeptides. Moreover, as more and moreglycopeptides are discovered to be useful for the treatment of a varietyof diseases, there is a need for methods that lower the cost of theirproduction. Further, there is also a need in the art to develop methodsof more efficiently producing recombinant glycopeptides for use indeveloping and improving glycopeptide therapeutics.

Glycosyltransferases are reviewed in general in International (PCT)Patent Application No. WO03/031464 (PCT/US02/32263), which isincorporated herein by reference in its entirety. One such particularglycosyltransferase that has utility in the development and productionof therapeutic glycopeptides is ST6GalNAcI. ST6GalNAcI, orGalNAcα2,6-sialyltransferase, catalyzes the transfer of sialic acid froma sialic acid donor to a sialic acid acceptor. Full length chickenST6GalNAcI enzyme, for example, is disclosed by Kurosawa et al. (1994, JBiol. Chem. 269:1402-1409). However, the identification of usefulmutants of this enzyme, having enhanced biological activity such asenhanced catalytic activity or enhanced stability, has not heretoforebeen reported.

In the past, there have been efforts to increase the availability ofrecombinant glycosyltransferases for the in vitro production ofglycopeptides. To date, a limited amount of work has been done withrespect to recombinant glycosyltransferases that may sometimes besuitable for small-scale production of oligosaccharides orglycopeptides. For example, Kurosawa et al. (1994, J Biol Chem. 269:1402-1409) describe a truncation mutant of chicken ST6GalNAcI lackingamino acid residues 1-232 of the full-length enzyme. A truncation ofmouse ST6GalNAcI was also reported by Kurosawa et al. (2000, J.Biochem., 127:845-854). However, for example, the truncated chickenenzyme described by Kurosawa et al. tacks the substrate specificity ofother ST6GalNAcI enzymes and lacks the activity required for“pharmaceutical-scale” processes and reactions, including the productionof glycopeptide therapeutics. Therefore, a need still exists forrecombinant glycosyltransferases having activity that is suitable for“pharmaceutical-scale” processes and reactions, including the productionof glycopeptide therapeutics. In particular, there is a need forrecombinant glycosyltransferases having favorable functional andstructural characteristics. Further, a need exists for efficient methodsof identification and characterization of recombinantglycosyltransferases, as well as for the production of suchglycosyltransferases. The present invention addresses and meets theseneeds.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated truncated ST6GalNAcIpolypeptide that lacks all or a portion of e.g., the ST6GalNAcI signaldomain, all or a portion of the ST6GalNAcI transmembrane domain, or allor a portion of the ST6GalNAcI stem domain; with the proviso that saidpolypeptide is not a chicken ST6GalNAcI polypeptide truncation mutantlacking amino acid residues 1-232. The truncated ST6GalNAcI polypeptidescan be e.g., a truncated human ST6GalNAcI, a truncated chickenST6GalNAcI, or a truncated mouse ST6GalNAcI.

In one embodiment, the truncated ST6GalNAcI polypeptide has at least 90%or 95% identity with a polypeptide selected from SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, Δ35 of the human sequence shown in FIG. 31, Δ72of the human sequence shown in FIG. 31, Δ109 of the human sequence shownin FIG. 31, Δ133 of the human sequence shown in FIG. 31, Δ170 of thehuman sequence shown in FIG. 31, Δ232 of the human sequence shown inFIG. 31, Δ272 of the human sequence shown in FIG. 31, SEQ ID NO:28, SEQID NO:30, SEQ ID NO:32, Δ225 of the chicken sequence shown in FIG. 31,SEQ ID NO: 18, Δ30 of the mouse sequence shown in FIG. 31, Δ51 of themouse sequence shown in FIG. 31, SEQ ID NO:22, SEQ ID NO:24 and Δ200 ofthe mouse sequence shown in FIG. 31. In another embodiment, the isolatedtruncated ST6GalNAcI polypeptide comprises an amino acid sequence of SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, Δ35 of the human sequence shownin FIG. 31, Δ72 of the human-sequence shown in FIG. 31 Δ109 of the humansequence shown in FIG. 31, Δ133 of the human sequence shown in FIG. 31,Δ170 of the human sequence shown in FIG. 31, Δ232 of the human sequenceshown in FIG. 31, Δ272 of the human sequence shown in FIG. 31, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, Δ225 of the chicken sequence shown inFIG. 31, SEQ ID NO: 18, Δ30 of the mouse sequence shown in FIG. 31, Δ51of the mouse sequence shown in FIG. 31, SEQ ID NO:22, SEQ ID NO:24 andΔ200 of the mouse sequence shown in FIG. 31.

The truncated ST6GalNAcI polypeptide can be a fusion protein andcomprise a tag polypeptide, such as, e.g., a maltose binding protein, ahistidine tag, a Factor IX tag, a glutathione-S-transferase tag, aFLAG-tag, and a starch binding domain tag.

In another aspect, the invention include isolated nucleic acid moleculesthat encode the truncated ST6GalNAcI polypeptides described above. Thenucleic acids can be operably linked to a promoter/regulatory sequenceor can be part of an expression vector. The invention also include hostcells that comprise expression vectors that encode the truncatedST6GalNAcI polypeptides described above. Such host cells can beeukaryotic or prokaryotic host cells. Eukaryotic cells include e.g.,mammalian cells, insect cells, and a fungal cells. Insect cells caninclude e.g., SF9 cells, SF9+ cells, Sf21 cells, HIGH FIVE cells, orDrosophila Schneider S2 cells. Preferred prokaryotic cells include e.g.,E. coli cells and B. subtilis cells. The invention also include methodsof using the host cells to produce truncated ST6GalNAcI polypeptides, bygrowing the recombinant host cells under conditions suitable forexpression of the truncated ST6GalNAcI polypeptide.

In another aspect, the present invention includes a method of catalyzingthe transfer of a sialic acid moiety to an acceptor moiety using thetruncated ST6GalNAcI polypeptides described above to mediate thecovalent linkage of said sialic acid moiety to said acceptor moiety,thereby catalyzing the transfer of a sialic acid moiety to an acceptormoiety.

In a further aspect, the invention provides a method of catalyzing thetransfer of a sialic acid moiety to an acceptor moiety by incubating thetruncated ST6GalNAcI polypeptides described above with acytidinemonophosphate-sialic acid (CMP-NAN) sialic acid donor and anasialo bovine submaxillary mucin acceptor moiety, wherein saidpolypeptide mediates the transfer of said sialic acid moiety from saidCMP-NAN sialic acid donor to said asialo bovine submaxillary mucinacceptor, thereby catalyzing the transfer of a sialic acid moiety to anacceptor moiety. In preferred embodiment, the acceptor is a polypeptideacceptor, such as e.g., erythropoietin, human growth hormone,granulocyte colony stimulating factor, interferons alpha, -beta, and-gamma, Factor IX, follicle stimulating hormone, interleukin-2,erythropoietin, anti-TNF-alpha, and a lysosomal hydrolase. In otherembodiments, the polypeptide acceptor is a glycopeptide. In a furtherpreferred embodiment, the sialic acid moiety comprises a polyethyleneglycol moiety. In a still further embodiment the method is carried outon a commercial scale to make commercial scale amounts of a sialylatedproduct, e.g., a sialylated glycoprotein or glycopeptide.

In another aspect, the invention provides an isolated truncated human orchicken ST6GalNAcI polypeptide that lacks all or a portion of theST6GalNAcI signal domain, with the proviso that said polypeptide is nota chicken ST6GalNAcI polypeptide truncation mutant lacking amino acidresidues 1-232. In other embodiments, the truncated human or chickenST6GalNAcI polypeptide can additionally lack all or a portion of theST6GalNAcI transmembrane domain or can lack all or a portion of theST6GalNAcI stem domain.

In some embodiments, the truncated human or chicken ST6GalNAcIpolypeptide includes an amino acid sequence with at least 90% or 95%identity to the following: SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,Δ35 of the human sequence shown in FIG. 31, Δ72 of the human sequenceshown in FIG. 31, Δ109 of the human sequence shown in FIG. 31, Δ133 ofthe human sequence shown in FIG. 31, Δ170 of the human sequence shown inFIG. 31, Δ232 of the human sequence shown in FIG. 31, Δ272 of the humansequence shown in FIG. 31, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, andΔ225 of the chicken sequence shown in FIG. 31. In a further embodiment,the truncated human or chicken ST6GalNAcI polypeptide includes an aminoacid sequence selected from the group consisting of SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, Δ35 of the human sequence shown in FIG. 31, Δ72 ofthe human sequence shown in FIG. 31, Δ109 of the human sequence shown inFIG. 31, Δ133 of the human sequence shown in FIG. 31, Δ170 of the humansequence shown in FIG. 31, Δ232 of the human sequence shown in FIG. 31,Δ272 of the human sequence shown in FIG. 31, SEQ ID NO:28, SEQ ID NO:30,SEQ ID NO:32, and Δ225 of the chicken sequence shown in FIG. 31.

The truncated human or chicken ST6GalNAcI polypeptide can be a fusionprotein and comprise a tag polypeptide, such as, e.g., a maltose bindingprotein, a histidine tag, a Factor IX tag, a glutathione-S-transferasetag, a FLAG-tag, and a starch binding domain tag.

In another aspect, the invention include isolated nucleic acid moleculesthat encode the truncated human or chicken ST6GalNAcI polypeptidesdescribed above. The nucleic acids can be operably linked to apromoter/regulatory sequence or can be part of an expression vector. Theinvention also includes host cells that comprise expression vectors thatencode the truncated human or chicken ST6GalNAcI polypeptides describedabove. Such host cells can be eukaryotic or prokaryotic host cells.Eukaryotic cells include, e.g., mammalian cells, insect cells, and afungal cells. Insect cells can include e.g., SF9 cells, SF9+ cells, Sf2cells, HIGH FIVE cells, or Drosophila Schneider S2 cells. Preferredprokaryotic cells include e.g., E. coli cells and B. subtilis cells. Theinvention also include methods of using the host cells to producetruncated human or chicken ST6GalNAcI polypeptides, by growing therecombinant host cells under conditions suitable for expression of thetruncated human or chicken ST6GalNAcI polypeptide.

In another aspect, the present invention includes a method of catalyzingthe transfer of a sialic acid moiety to an acceptor moiety using thetruncated human or chicken ST6GalNAcI polypeptides described above tomediate the covalent linkage of said sialic acid moiety to said acceptormoiety, thereby catalyzing the transfer of a sialic acid moiety to anacceptor moiety.

In a further aspect, the invention provides a method of catalyzing thetransfer of a sialic acid moiety to an acceptor moiety by incubating thetruncated human or chicken ST6GalNAcI polypeptides described above witha cytidinemonophosphate-sialic acid (CMP-NAN) sialic acid donor and anasialo bovine submaxillary mucin acceptor moiety, wherein saidpolypeptide mediates the transfer of said sialic acid moiety from saidCMP-NAN sialic acid donor to said asialo bovine submaxillary mucinacceptor, thereby catalyzing the transfer of a sialic acid moiety to anacceptor moiety. In preferred embodiment, the acceptor is a polypeptideacceptor, such as e.g., erythropoietin, human growth hormone,granulocyte colony stimulating factor, interferons alpha, -beta, and-gamma, Factor IX, follicle stimulating hormone, interleukin-2,erythropoietin, anti-TNF-alpha, and a lysosomal hydrolase. In otherembodiments, the polypeptide acceptor is a glycopeptide. In a furtherpreferred embodiment, the sialic acid moiety comprises a polyethyleneglycol moiety. In a still further embodiment the method is carried outon a commercial scale to make commercial scale amounts of a sialylatedproduct, e.g., a sialylated glycoprotein or glycopeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

For purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a diagram illustrating the location of restriction enzymecleavage sites within the mouse ST6GalNAcI truncation mutants Δ31, Δ51,Δ126, Δ185, and Δ200 (referenced as D32, E52, S127, S186 and S201 in theillustration, respectively). The figure also illustrates the respectivesize, in bp, of each construct.

FIG. 2 is an image of an electrophoretic gel DNA fragments of 1488 bp,1428 bp, 1203 bp, 1026 bp, and 981 bp, corresponding respectively toD32, E52, S127, S186, and S201 of N-terminal amino acid truncatedST6GalNAcI nucleic acids. Lane 1, bp ladder; lane 2, D32; lane 3, E52;lane 4, S127; lane 5, S186; lane 6, S201.

FIG. 3 is an image of an electrophoretic gel containing DNA fromrestriction enzyme digestions using endonucleases BamHI/XhoI for E52,S127, S186 and S201 mouse ST6GalNAcI constructs and HindIII/XhoI for theD32 mouse ST6GalNAcI construct. DNA fragments of approximately 1.5 to1.0 Kb correspond to different truncation mutants of ST6GalNAcI. Thelarger fragment visible near 6.0 Kb is pCWin2-MBP. Lane 1, bp ladder;upper lanes 2-4, E52; upper lanes 5-7, S127; upper lanes 8-10, S186;upper lanes 11-12, S201; lower lanes 2-5, D32, lower lanes 7-9,MBP-pCWin2.

FIG. 4 is an image of an electrophoretic gel illustrating the results ofthe screening of recombinant colonies DH5α/pCWin2-MBP-ST6GalNAcI, usingHindIII/XhoI restriction enzymes to digest the D32 construct andBamHI/XhoI to digest the constructs E52, S127, S186 and S201. All 4colonies from each truncation (numbered 1 through 4) released a fragmentof approximately 1.5 to 1.0 Kb corresponding respectively to D32, E52,S127, S186 and S201 of ST6GalNAcI and a larger fragment around 6.0 Kbrepresenting the MBP-pCWin2 vector. Lane 1, bp ladder. Upper lanes 1, 3,5, 7 and 9, uncut D32/vector; upper lanes 11, 13, 15, uncut E52/vector;upper lanes 17 and 19, uncut S127/vector; upper lanes 2, 4, 6, and 8,cut D32; upper lanes 10, 12 and 14, cut E52; upper lanes 16 and 18, cutS127; lower lanes 1, 3 and 5, uncut S127; lower lanes 7, 9 and 11, uncutS186; lower lanes 13, 15, 17 and 19, uncut S201; lower lanes 2 and 4,cut S127; lower lanes 6, 8, 10 and 12, cut S186; lower lanes 14, 16 and18, cut S201.

FIG. 5 is an image of an electrophoretic gel illustrating restrictiondigestion analysis on plasmid DNA isolated from colonies #1 thru #2 ofeach construct DH5α/pCWin2-MBP-ST6GalNAcI. Plasmid DNA was doubledigested with NdeI/HindIII enzymes. All colonies except for the1)₃₂-containing colonies released a single band around 2.5 Kb (D32released two fragments) which is indicative of the MBP-ST6GalNAcIinsert, while the larger expected band around 5.0 Kb corresponds to thepCWin2 vector. M=bp ladder. Lanes 1, 3=D32; 5, 7=E52; 11, 22=S127; 12,14=S186 and 16, 18 S201, and all contain uncut DNA. Lanes 2, 4=D32; 6,8=E52; 10, 21=S127; 13, 15 S186 and 17, 19=S201, and all containdigested DNA.

FIG. 6 is an image of an electrophoretic gel illustrating diagnosticrestriction enzyme digestion of construct JM109/pCWin2-MBP-ST6GalNAcI,using NdeI/XhoI restriction enzymes. All colonies, with the exception ofD32, released a fragment around 2.5 Kb corresponding to truncatedMBP-ST6GalNAcI fusion protein (D32 released two fragments). Fragments at5.0 Kb correspond to the pCWin2 vector. MW=bp ladder. Lanes 1, 3=D32;lanes 5, 7=E52; lanes 9, 11=S127; lanes 13, 15=S186; lanes 17 and19=S201, and all contain uncut DNA. Lanes 2, 4=D32; lanes 6, 8=E52;lanes 10, 12=S127; lanes 14, 16=S186; lanes 18 and 20=S201 and allcontain digested DNA.

FIG. 7 is an image of an electrophoretic protein gel illustrating thepresence of polypeptides corresponding to the expected size of therespective mouse ST6GalNAcI truncation mutants present in cell lysateand inclusion bodies for the cells harboring the respective DNAconstructs. Lane MW contains a MW marker. Each “lane 1” contains D32,each “lane 2” contains E52, each “lane 3,” contains S127, each “lane 4”contains S186, and each “lane 5” contains S201.

FIG. 8 is an image of an electrophoretic protein gel illustrating theexpression of truncated forms of mouse ST6GalNAcI as an MBP fusionprotein in lysates and inclusion bodies obtained from JM109 cells. LaneMW contains a MW marker. Each “lane I” contains D32, each “lane 2”contains E52, each “lane 3” contains S127, each “lane 4” contains S186,and each “lane 5” contains S201.

FIG. 9 is an image of an electrophoretic protein gel illustrating theexpression of MBP-ST6GalNAcI in JM109 and W3110/pCWin2 MBP-ST6GalNAcIconstructs S186 and S201. Lane MW contains a MW marker Lane 1 containsS186 from w3110 #11, 1.0 mg.ml; lane 2 contains S8186 from w3110 #11,0.1 mg/ml; lane 3 contains S186 from JM109 #11, 1.0 mg/ml; lane 4contains S186 from JM109 #11, 0.1 mg/ml; lane 5 contains S201 from w3110#8, 1.0 mg.ml; lane 6 contains S201 from w3110 #8, 0.1 mg/ml; lane 7contains S201 from JM109 #8, 1.0 mg/ml; lane 8 contains S201 from JM109#8, 0.1 mg/ml.

FIG. 10 is an image of a mass spectrometric depiction of the transfer ofsialic acid to a GalNAc−O-G-CSF acceptor by bacterially-isolated,refolded ST6GalNAcI construct S201. Panel A illustrates a sample takenat 24 hours, Panel B illustrates a sample taken at 48 hours, Panel Cillustrates a sample taken at 2 days, and Panel D illustrates a sampletaken at 5 days.

FIG. 11 is an image of an electrophoretic gel confirming the humanST6GalNAcI inserts of EST clones by restriction enzymatic digestion.Lanes 1-3, clone#1-3 of EST clone#4816713 digested by EcoR I; Lane 4,1-Kb ladder; lanes 5-6, clone#1-3 of EST clone#6300955 digested by EcoRI and Xho I.

FIG. 11 is an image of an electrophoretic gel confirming the humanST6GalNAcI inserts of EST clones by restriction enzymatic digestion.Lanes 1-3, clone#1-3 of EST clone#4816713 digested by EcoR I Lane 4,1-Kb ladder, lanes 5-6, clone#1-3 of EST clone#6300955 digested by EcoRI and Xho I.

FIG. 12 is a diagram illustrating an alignment of cDNA sequences of the#4816713 and clone#6300955 human ST6GalNAcI EST clones clones.

FIG. 13 is an image of an electrophoretic gel illustrating the EcoRIrestriction digestion of pCR-hST6-N and pCR-hST6-C of all six humanST6GalNAcI clones containing the correct sizes cDNA insert. Lanes 1-6contain a restriction digest of six pCR-hST6-N clones; lanes 7-12contain a restriction digest of six pCR-hST6-C clones.

FIG. 14 is an image of an electrophoretic gel illustrating restrictionenzyme digestions of pcDNA3.1-hST6GalNAcI. Panel A: Lane 1, 1-Kb ladder;lanes 2-7, pcDNA3.1-hST6GalNAcI clone #1-6. Panel B: illustration ofrestriction enzyme map of pcDNA3.1-hST6GalNAcI.

FIG. 15 illustrates the nucleotide and amino acid sequences ofpcDNA3.1(+)-hST6GalNAcI-NIC#1.

FIG. 16 is a cartoon depicting the domain structures and the varioustruncation mutants of human ST6GalNAcI.

FIG. 17A is a plasmid map of the pAcGP67-B baculovirus transfer vector.

FIG. 17B is a map illustrating the cloning site of the pAcGP67-Bbaculovirus transfer vector.

FIG. 18 is a graph depicting ST6GalNAcI activities in Sf9 cell culturemedium for K36, K125 and S258 human ST6GalNAcI constructs, and forpTS103.

FIG. 19A illustrates the nucleotide and amino acid sequences of mouseST6GalNAcI from pTS103.

FIG. 19B is a cartoon depicting the domain structures and the varioustruncation mutants of mouse ST6GalNAcI.

FIG. 20A is a plasmid map of the pFastBac1 vector.

FIG. 20B is a map of the polycloning sites of the pFastBac-1-gp vector.

FIG. 21 is an image of an electrophoretic gel illustrating plasmid DNAsubjected to EcoRI and XhoI restriction digestions to release mouseST6GalNAcI DNA inserts from pFastBac-1-gp-mST6GalNAcI. Lanes 1-4,clones# 1-4 of S127 truncation mutant; lanes 5-8, clones #1-4 of S186truncation mutant; lane 9, 1 kb ladder.

FIG. 22A is a diagram of the primer pairs on the pFastBac-1 bacmid.

FIG. 22B is an image of an electrophoretic gel illustrating PCRscreening of mouse ST6GalNAcI cDNA in the bacmid DNA. Electrophoresis ofthe PCR products was conducted on a 1% agarose gel. Lane 1, 1-kb ladder;lanes 2-9, clones 1-8 of the recombinant bacmid DNA.

FIG. 23 is an image of an electrophoretic gel illustrating analysis ofmouse ST6GalNAcI bacmid DNA on a 1% agarose gel. Lane 1, 1-kb ladder;lane 2, S186#3; lane 3, S186#4; lane 4, S127#5; lane 5, S12746.

FIG. 24 is a graph depicting ST6GalNAcI activities in Sf9 cell culturemedium for mouse ST6GalNAcI constructs S127#5, S127#6, S186#3, S186#4,and for the pTS103 plasmid.

FIG. 25 is a table depicting the titer calculations of viral stocks foruse in the screening of chicken ST6GalNAcI truncated mutant constructs.

FIG. 26 illustrates the full-length nucleic acid sequence of chickenST6GalNAcI.

FIG. 27 illustrates the amino acid sequence as translated based on theDNA sequence of FIG. 26.

FIG. 28 illustrates the nucleic acid sequence of full length chickenST6GalNAcI as set forth in GenBank Accession Number X74946.

FIG. 29 illustrates the nucleic acid sequence of K232 chickenST6GalNAcI.

FIG. 30 illustrates the amino acid sequence of K232 truncated chickenST6GalNAcI.

FIG. 31 is a sequence comparison of human, mouse and chicken ST6GalNAcIamino acid sequences. The starting residues for Δ48, Δ152, Δ225 and Δ232mutants—amino acids Q49, V153, L226 and T233, respectively—aresurrounded by boxes.

FIG. 32 is an image of an electrophoretic protein gel illustrating thesialylPEGylation of G-CSF by Δ48, Δ152, Δ225 mutant ST6GalNAcI enzymes.Lane 1, MW marker; lane 2, G-CSF sialylPEGylated with Δ48(MOI=0.8, 35.6U/L); lane 3, G-CSF sialylPEGylated with Δ152 (MOI=1.43, 39.5 U/L); lane4, G-CSF sialylPEGylated with Δ232 (MOI=0.531, 0 U/L); lane 5, G-CSFsialylPEGylated with K232 VS4-001 ST6GalNAcI (supernatant); lane 6,G-CSF sialylPEGylated with K232 VS4-001 ST6GalNAcI (purified); lane 7,G-CSF sialylPEGylated with Δ48(MOI=0.2, 35.4 U/L); lane 8, G-CSFsialylPEGylated with Δ152 (MOI=0.356, 39.9 U/L); lane 9, G-CSFsialylPEGylated with Δ232 (MOI=0.133, 0 U/L); lane 10, G-CSFsialylPEGylated with Δ232 (MOI=0.133, 0 U/L); lane 11, G-CSFsialylPEGylated with K232 VS4-001 ST6GalNAcI (purified); lane 12, MWmarker.

FIG. 33 is an image of an electrophoretic protein gel illustrating thesialylPEGylation of G-CSF by Δ48, Δ152, Δ225 mutant ST6GalNAcI enzymes.Lane 1, MW marker; lane 2, G-CSF sialylPEGylated with Δ48(MOI=0.8, 35.6U/L); lane 3, G-CSF sialylPEGylated with Δ48(MOI=0.2, 35.4 U/L); lane 4,G-CSF sialylPEGylated with Δ152 (MOI=1.43, 39.5 U/L); lane 5, G-CSFsialylPEGylated with Δ152 (MOI=0.356, 39.9 U/L); lane 6, G-CSFsialylPEGylated with Δ225 (27.9 U/L); lane 7, G-CSF sialylPEGylated withK232 VS4-001 ST6GalNAcI (purified); lane 8, MW marker.

FIG. 34 provides full length amino acid sequences for A) humanST6GalNAcI and for B) chicken ST6GalNAcI, and C) a sequence of the mouseST6GalNAcI protein beginning at residue 32 of the native mouse protein.

FIG. 35 provides a schematic of a number of preferred human ST6GalNAcItruncation mutants.

FIG. 36 shows a schematic of MBP fusion proteins including the humanST6GalNAcI truncation mutants.

FIG. 37 shows the position of paired and unpaired cysteine residues inthe human ST6GalNAcI protein. Single and double cysteine substitutionare also shown, e.g., C280S, C362S, C362T, (C280S+C362S), and(C280S+C362T).

FIG. 38 shows ST6GalNAcI activities of human turncated proteins.Activities were determined in samples obtained from a bacculoviralsystem.

FIG. 39 shows amino acid sequence alignments of three ST6GalNAcIenzymes: Human, chicken and mouse. The original human enzyme truncationwas at Δ35 (K36) position right after membrane spanning region. Inaddition to earlier human ST6GalNAcI truncations, here 6 more humanenzyme truncations were designed and generated. The first one Δ72 (T73)was based on protease cleavage and the rest were designed based onhomologous regions among the three or two enzymes. The last truncationΔ272 (G273) was analogous to early chicken ST6GalNAcI truncation. Thearrows indicate the truncations in the human protein. The figure alsoshows an alignment of the human sequence with the mouse and chickenproteins and identifies identical and conserved amino acid residuesbetween the proteins.

FIG. 40 shows schematic of a three way fusion between a gp67 secretionpeptide, an ST6GalNAcI coding sequence, and an SBD coding sequence. Thefusion proteins were expressed in baculovirus, purified on acyclodextrin column, and assayed for enzymatic activity.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods of the present invention encompasstruncation mutants of human ST6GalNAcI, mouse ST6GalNAcI and chickenST6GalNAcI, isolated nucleic acids encoding these proteins, and methodsof their use. ST6GalNAcI polypeptides catalyze the transfer of sialicacid from a sialic acid donor to a sialic acid acceptor.

The glycosyltransferase ST6GalNAcI is an essential reagent forglycosylation of therapeutic glycopeptides. Additionally, ST6GalNAcI isan important reagent for research and development of therapeuticallyimportant glycopeptides and oligosaccharide therapeutics. ST6GalNAcI istypically isolated and purified from natural sources, or from tediousand costly in vitro and recombinant sources. The present inventionprovides compositions and methods relating to simplified and morecost-effective methods of production of ST6GalNAcI enzymes. Inparticular, the present invention provides compositions and methodsrelating to truncated ST6GalNAcI enzymes that have improved and usefulproperties in comparison to their full-length enzyme counterparts.

Truncated glycosyltransferase enzymes of the present invention areuseful for in vivo and in vitro preparation of glycosylated peptides, aswell as for the production of oligosaccharides containing the specificglycosyl residues that can be transferred by the truncatedglycosyltransferase enzymes of the present invention. This is because itis shown for the first time herein that truncated forms of ST6GalNAcIpolypeptides possess biological activities comparable to, and in someinstances, in excess of their full-length polypeptide counterparts. Thepresent application also discloses that such truncation mutants not onlypossess biological activity, but also that the truncation mutants mayhave enhanced properties of solubility, stability and resistance toproteolytic degradation.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein.

Certain abbreviations are used herein as are common in the art, such as:“Ac” for acetyl, “Glc” for glucose; “Glc” for glucosamine; “GlcA forglucuronic acid; “IdoA” for iduronic acid; “GlcNAc” forN-acetylglucosamine; “NAN” or “sialic acid” or “SA” for N-acetylneuraminic acid; “UDP” for uridine diphosphate; “CMP” for cytidinemonophosphate.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anticodon regionof a transfer RNA molecule during translation of the mRNA molecule orwhich encode a stop codon. The coding region may thus include nucleotideresidues corresponding to amino acid residues which are not present inthe mature protein encoded by the mRNA molecule (e.g., amino acidresidues in a protein export signal sequence).

An “affinity tag” is a peptide or polypeptide that may be genetically orchemically fused to a second polypeptide for the purposes ofpurification, isolation, targeting, trafficking, or identification ofthe second polypeptide. The “genetic” attachment of an affinity tag to asecond protein may be effected by cloning a nucleic acid encoding theaffinity tag adjacent to a nucleic acid encoding a second protein in anucleic acid vector.

As used herein, the term “glycosyltransferase,” refers to anyenzyme/protein that has the ability to transfer a donor sugar to anacceptor moiety.

A “sugar nucleotide-generating enzyme” is an enzyme that has the abilityto produce a sugar nucleotide. Sugar nucleotides are known in the art,and include, but are not limited to, such moieties as UDP-Gal,UDP-GalNAc, and CMP-NAN.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or cDNA fragment produced by PCP or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.However, the terms “nucleic acid” and “polynucleotide” are usedinterchangeably herein.

The term “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

Conventional notation is used herein to describe nucleic acid sequences:the left-hand end of a single-stranded nucleic acid sequence is the 5′end; the left-hand direction of a double-stranded nucleic acid sequenceis referred to as the 5′-direction.

A first defined nucleic acid sequence is said to be “immediatelyadjacent to” a second defined nucleic acid sequence when, for example,the last nucleotide of the first nucleic acid sequence is chemicallybonded to the first nucleotide of the second nucleic acid sequencethrough a phosphodiester bond. Conversely, a first defined nucleic acidsequence is also said to be “immediately adjacent to” a second definednucleic acid sequence when, for example, the first nucleotide of thefirst nucleic acid sequence is chemically bonded to the last nucleotideof the second nucleic acid sequence through a phosphodiester bond.

A first defined polypeptide sequence is said to be “immediately adjacentto” a second defined polypeptide sequence when, for example, the lastamino acid of the first polypeptide sequence is chemically bonded to thefirst amino acid of the second polypeptide sequence through a peptidebond. Conversely, a first defined polypeptide sequence is said to be“immediately adjacent to” a second defined polypeptide sequence when,for example, the first amino acid of the first polypeptide sequence ischemically bonded to the last amino acid of the second polypeptidesequence through a peptide bond.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

As used herein, the term “percent identity” is used synonymously with“homology.” The determination of percent identity between two nucleotideor amino acid sequences can be accomplished using a mathematicalalgorithm. For example, a mathematical algorithm useful for comparingtwo sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl.Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993,Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990, J. Mol.Biol. 215:403-410), and can be accessed, for example, at the BLAST siteof the National Center for Biotechnology Information (NCBI) world wideweb site at the National Library of Medicine (NLM) at the NationalInstitutes of Health (NIH). BLAST nucleotide searches can be performedwith the NBLAST program (designated “blastn” at the NCBI web site),using the following parameters: gap penalty 5; gap extension penalty=2;mismatch penalty=3; match reward=1; expectation value 10.0; and wordsize=11 to obtain nucleotide sequences homologous to a nucleic aciddescribed herein. BLAST protein searches can be performed with theXBLAST program (designated “blastn” at the NCBI web site) or the NCBI“blastp” program, using the following parameters: expectation value10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologousto a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used as available on the website of theNational Center for Biotechnology Information of the National Library ofMedicine at the National Institutes of Health.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer. A“polypeptide,” as the term is used herein, therefore refers to any sizepolymer of amino acid residues, provided that the polymer contains atleast two amino acid residues.

The term “protein” typically refers to large peptides, also referred toherein as “polypeptides.” The term “peptide” typically refers to shortpolypeptides. However, the terms “peptide,” “protein” and “polypeptide”are used interchangeably herein. For example, the term “peptide” mayrefer to an amino acid polymer of three amino acids, as well as an aminoacid polymer of several hundred amino acids.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

A “therapeutic peptide” as the term is used herein refers to any peptidethat is useful to treat a disease state or to improve the overall healthof a living organism. A therapeutic peptide may effect such changes in aliving organism when administered alone, or when used to improve thetherapeutic capacity of another substance. The term “therapeuticpeptide” is used interchangeably herein with the terms “therapeuticpolypeptide” and “therapeutic protein.”

A “reagent peptide” as the term is used herein refers to any peptidethat is useful in food biochemistry, bioremediation, production of smallmolecule therapeutics, and even in the production of therapeuticpeptides. Typically, reagent peptides are enzymes capable of catalyzinga reaction to produce a product useful in any of the aforementionedareas. The term “reagent peptide” is used interchangeably herein withthe terms “reagent polypeptide” and “reagent protein.”

A “glycopeptide” as the term is used herein refers to a peptide havingat least one carbohydrate moiety covalently linked thereto. It will beunderstood that a glycopeptide may be a “therapeutic glycopeptide,” asdescribed above. The term “glycopeptide” is used interchangeably hereinwith the terms “glycopolypeptide” and “glycoprotein.”

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear nucleic acids, nucleic acidsassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinant nucleicacid comprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant nucleic acid.

A “multiple cloning site” as the term is used herein is a region of anucleic acid vector that contains more than one sequence of nucleotidesthat is recognized by at least one restriction enzyme.

An “antibiotic resistance marker” as the term is used herein refers to asequence of nucleotides that encodes a protein which, when expressed ina living cell, confers to that cell the ability to live and grow in thepresence of an antibiotic.

As used herein, the term “ST6GalNAcI” refers toN-acetylgalactosamine-α2,6-sialyltransferase I.

As the term is used herein, a “truncated” form of a peptide refers to apeptide that is lacking one or more amino acid residues as compared tothe full-length amino acid sequence of the peptide. For example, thepeptide “NH2-Ala-Glu-Lys-Leu-COOH” is an N-terminally truncated form ofthe frill-length peptide “NH2-Gly-Ala-Glu-Lys-Leu-COOH.” The terms“truncated form” and “truncation mutant” are used interchangeablyherein. By way of a non-limiting example, a truncated peptide is aST6GalNAcI polypeptide comprising an active domain, a stem domain, atransmembrane domain, and a signal domain, wherein the signal domain islacking a single N-terminal amino acid residue as compared to the fulllength ST6GalNAcI.

The term “saccharide” refers in general to any carbohydrate, a chemicalentity with the most basic structure of (CH₂O)_(n). Saccharides vary incomplexity, and may also includes nucleic acid, amino acid, or virtuallyany other chemical moiety existing in biological systems

“Monosaccharide” refers to a single unit of carbohydrate of a definedidentity.

“Oligosaccharide” refers to a molecule consisting of several units ofcarbohydrates of defined identity. Typically, saccharide sequencesbetween 2-20 units may be referred to as oligosaccharides.

“Polysaccharide” refers to a molecule consisting of many units ofcarbohydrates of defined identity. However, any saccharide of two ormore units may correctly be considered a polysaccharide.

As used herein, a saccharide “donor” is a moiety that can provide asaccharide to a glycosyltransferase so that the glycosyltransferase maytransfer the saccharide to a saccharide acceptor. By way of anon-limiting example, a GalNAc donor may be UDP-GalNAc.

As used herein, a saccharide “acceptor” is a moiety that can accept asaccharide from a saccharide donor. A glycosyltransferase can covalentlycouple a saccharide to a saccharide acceptor. By way of a non-limitingexample, G-CSF may be a GalNAc acceptor, and a GalNAc moiety may becovalently coupled to a GalNAc acceptor by way of a GalNAc−transferase.

An oligosaccharide with a “defined size” is one which consists of anidentifiable number of monosaccharide units. For example, anoligosaccharide consisting of 10 monosaccharide units is one which mayconsist of 10 identical monosaccharide units or 5 monosaccharide unitsof a first identity and 5 monosaccharide units of a second identity.Further, an oligosaccharide of defined size that consists ofmonosaccharide units of heterogeneous identity may have themonosaccharide units in any order from beginning to end of theoligosaccharide.

An oligosaccharide of “random size” is one which may be synthesizedusing methods that do not provide oligosaccharide products of definedsize. For example, a method of oligosaccharide synthesis may provideoligosaccharides that range from two monosaccharide units to twenty-twosaccharide units, including any or all lengths in between.

“Commercial scale” refers to gram scale production of a productsaccharide, or glycoprotein, or glycopeptide in a single reaction. Inpreferred embodiments, commercial scale refers to production of greaterthan about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 grams.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-NeusAc or 9-O-acetyl-NeuSAc,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

A “method of remodeling a protein, a peptide, a glycoprotein, or aglycopeptide” as used herein, refers to addition of a sugar residue to aprotein, a peptide, a glycoprotein, or a glycopeptide using aglycosyltransferase. In a preferred embodiment, the sugar residue iscovalently attached to a PEG molecule.

An “unpaired cysteine residue” as used herein, refers to a cysteineresidue, which in a correctly folded protein (i.e., a protein withbiological activity), does not form a disulfide bind with anothercysteine residue.

An “insoluble glycosyltransferase” refers to a glycosyltransferase thatis expressed in bacterial inclusion bodies. Insolubleglycosyltransferases are typically solubilized or denatured using e.g.,detergents or chaotropic agents or some combination. “Refolding” refersto a process of restoring the structure of a biologically activeglycosyltransferase to a glycosyltransferase that has been solubilizedor denatured. Thus, a refolding buffer, refers to a buffer that enhancesor accelerates refolding of a glycosyltransferase.

A “redox couple” refers to mixtures of reduced and oxidized thiolreagents and include reduced and oxidized glutathione (GSH/GSSG),cysteine/cystine-cysteamine/cystamine, DTT/GSSG, and DTE/GSSG. (See,e.g., Clark, Cur. Op. Biotech. 12:202-207 (2001)).

The term “contacting” is used herein interchangeably with the following:combined with, added to, mixed with, passed over, incubated with, flowedover, etc.

The term “PEG” refers to poly(ethylene glycol). PEG is an exemplarypolymer that has been conjugated to peptides. The use of PEG toderivatize peptide therapeutics has been demonstrated to reduce theimmunogenicity of the peptides and prolong the clearance time from thecirculation. For example, U.S. Pat. No. 4,179,337 (Davis et al.)concerns non-immunogenic peptides, such as enzymes and peptide hormonescoupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10and 100 moles of polymer are used per mole peptide and at least 15% ofthe physiological activity is maintained.

The term “specific activity” as used herein refers to the catalyticactivity of an enzyme, e.g., a recombinant glycosyltransferase fissionprotein of the present invention, and may be expressed in activityunits. As used herein, one activity unit catalyzes the formation of 1μmol of product per minute at a given temperature (e.g., at 37° C.) andpH value (e.g., at pH 7.5). Thus, 10 units of an enzyme is a catalyticamount of that enzyme where 10 μmol of substrate are converted to 10μmol of product in one minute at a temperature of, e.g., 37° C. and a pHvalue of, e.g., 7.5.

“N-linked” oligosaccharides are those oligosaccharides that are linkedto a peptide backbone through asparagine, by way of anasparagine-N-acetylglucosamine linkage. N-linked oligosaccharides arealso called “N-glycans.” All N-linked oligosaccharides have a commonpentasaccharide core of Man₃GlcNAc₂. They differ in the presence of, andin the number of branches (also called antennae) of peripheral sugarssuch as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucoseand sialic acid. Optionally, this structure may also contain a corefucose molecule and/or a xylose molecule.

“O-linked” oligosaccharides are those oligosaccharides that are linkedto a peptide backbone through threonine, serine, hydroxyproline,tyrosine, or other hydroxy-containing amino acids.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 60%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor substrates for aparticular glycosyltransferase are glycosylated.

A “fusion protein” refers to a protein comprising amino acid sequencesthat are in addition to, in place of, less than, and/or different fromthe amino acid sequences encoding the original or native full-lengthprotein or subsequences thereof.

A “stem region” with reference to glycosyltransferases refers to aprotein domain, or a subsequence thereof, which in the nativeglycosyltransferases is located adjacent to the trans-membrane domain,and has been reported to function as a retention signal to maintain theglycosyltransferase in the Golgi apparatus and as a site of proteolyticcleavage. Stem regions generally start with the first hydrophilic aminoacid following the hydrophobic transmembrane domain and end at thecatalytic domain, or in some cases the first cysteine residue followingthe transmembrane domain. Exemplary stem regions include, but is notlimited to, the stem region of eukaryotic ST6GalNAcI, amino acidresidues from about 30 to about 207 (see e.g., the murine enzyme), aminoacids 35-278 for the human enzyme or amino acids 37-253 for the chickenenzyme; the stem region of mammalian GalNAcT2, amino acid residues fromabout 71 to about 129 (see e.g., the rat enzyme).

A “catalytic domain” refers to a protein domain, or a subsequencethereof, that catalyzes an enzymatic reaction performed by the enzyme.For example, a catalytic domain of a sialyltransferase will include asubsequence of the sialyltransferase sufficient to transfer a sialicacid residue from a donor to an acceptor saccharide. A catalytic domaincan include an entire enzyme, a subsequence thereof, or can includeadditional amino acid sequences that are not attached to the enzyme, ora subsequence thereof, as found in nature.

The term “isolated” refers to material that is substantially oressentially free from components which interfere with the activity of anenzyme. For a saccharide, protein, or nucleic acid of the invention, theterm “isolated” refers to material that is substantially or essentiallyfree from components which normally accompany the material as found inits native state. Typically, an isolated saccharide, protein, or nucleicacid of the invention is at least about 80% pure, usually at least about90%, and preferably at least about 95% pure as measured by bandintensity on a silver stained gel or other method for determiningpurity. Purity or homogeneity can be indicated by a number of means wellknown in the art. For example, a protein or nucleic acid in a sample canbe resolved by polyacrylamide gel electrophoresis, and then the proteinor nucleic acid can be visualized by staining. For certain purposes highresolution of the protein or nucleic acid may be desirable and HPLC or asimilar means for purification, for example, may be utilized.

DESCRIPTION I. Isolated Nucleic Acids A. Generally

Exemplified herein are various truncation mutants of mammalianST6GalNAcI and chicken ST6GalNAcI. However, the present invention shouldnot be construed to cover a chicken ST6GalNAcI truncation mutantpolypeptide lacking amino acid residues 1-232.

Full-length ST6GalNAcI nucleic acids encode polypeptides that have adomain structure similar to other glycosyltransferases, including anN-terminal signal domain, a transmembrane domain, a stem domain, and anactive domain, wherein the active domain may comprise the majority ofthe amino acid sequence of such polypeptides. As will be understood byone of skill in the art, the presence of domain structure(s) extraneousto the active domain of recombinant ST6GalNAcI polypeptides may have anegative effect on the solubility, stability and activity of thepolypeptide in an aqueous or in vitro environment. For example, whilenot wishing to be bound by any particular theory, the presence of ahydrophobic transmembrane domain on a recombinant ST6GalNAcI polypeptideused in an in vitro reaction mixture may render the polypeptide lesssoluble than a recombinant ST6GalNAcI polypeptide without a hydryophobictransmembrane domain, and further, may even decrease the enzymaticactivity of the polypeptide by affecting or destabilizing the foldedstructure.

Therefore, it is desirable to produce recombinant ST6GalNAcI nucleicacids that encode ST6GalNAcI that is shorter than full-lengthST6GalNAcI, for the purpose of enhancing the activity, stability and/orutility of ST6GalNAcI polypeptides. The present invention provides suchmodified forms of ST6GalNAcI More particularly, the present inventionprovides isolated nucleic acids encoding such truncated polypeptides.

Nucleic acids of the present invention encode truncated forms ofST66GalNAcI polypeptides, as described in greater detail elsewhereherein A truncated ST6GalNAcI polypeptide encoded by a nucleic acid ofthe present invention, also referred to herein as a “truncation mutant,”may be truncated in various ways, as would be understood by the skilledartisan. Examples of truncated polypeptides encoded by a nucleic acid ofthe present invention include, but are not limited to, a polypeptidelacking a single N-terminal residue, a polypeptide lacking a singleC-terminal residue, a polypeptide lacking both an single N-terminalresidue and a single C-terminal residue, a polypeptide lacking acontiguous sequence of residues from the N-terminus, a polypeptidelacking a contiguous sequence of residues from the C-terminus, and anycombinations thereof.

Therefore, it will be understood, based on the disclosure set forthherein, that truncations of nucleic acids encoding ST6GalNAcIpolypeptides may be made for numerous reasons. In one embodiment of theinvention, a truncation may be made in order to remove part or all ofthe nucleic acid sequence encoding the signal peptide domain of anST6GalNAcI.

In another embodiment of the invention, a truncation may be made inorder to remove part or all of a nucleic acid sequence encoding atransmembrane domain of an ST6GalNAcI. By way of a non-limiting example,removal of a part or all of a nucleic acid sequence encoding atransmembrane domain may increase the solubility or stability of theencoded ST6GalNAcI polypeptide and/or may increase the level ofexpression of the encoded polypeptide.

In yet another embodiment of the invention, a truncation may be made inorder to remove part or all of a nucleic acid sequence encoding a stemdomain of an ST6GalNAcI. By way of a non-limiting example, removal of apart or all of a nucleic acid sequence encoding a stem domain mayincrease the solubility or stability of the encoded ST6GalNAcIpolypeptide and/or may increase the level of expression of the encodedpolypeptide.

The skilled artisan, when armed with the disclosure set forth herein,would understand how to design and create a truncation mutant ofST6GalNAcI as set forth in detail elsewhere herein. In one aspect of theinvention, the nucleic acid residue at which a truncation is made may bea highly-conserved residue. In another aspect of the invention, thenucleic acid residue at which a truncation is made may be selected suchthat the encoded polypeptide has a new N-terminal amino acid residuethat will aid in the purification of the expressed polypeptide.

B. ST6GalNAcI Isolated Nucleic Acids

The present invention features nucleic acids encoding smaller thanfull-length ST6GalNAcI. That is, the present invention features anucleic acid encoding a truncated ST6GalNAcI polypeptide, provided thepolypeptide expressed by the nucleic acid retains the biologicalactivity of the full-length protein. In one aspect of the invention, atruncated polypeptide is a mammalian truncated ST6GalNAcI polypeptide.In another aspect of the invention, a truncated polypeptide is a humantruncated ST6GalNAcI polypeptide. In yet another aspect of theinvention, a truncated polypeptide is a mouse truncated ST6GalNAcIpolypeptide. In still another aspect of the invention, a truncatedpolypeptide is a chicken truncated ST6GalNAcI polypeptide.

As would be understood by the skilled artisan, a nucleic acid encoding afull-length ST6GalNAcI may contain a nucleic acid sequence encoding oneor more identifyable polypeptide domains in addition to the “activedomain,” the domain primarily responsible for the catalytic activity, ofST6GalNAcI. This is because it is known in that art that a full-lengthST6GalNAcI polypeptide contains a signal domain, a transmembrane domain,and a stem domain, in addition to an active domain. Accordingly, anucleic acid encoding a full-length ST6GalNAcI may encode a polypeptidethat has a signal domain at the amino-terminus of the polypeptide,followed by a transmembrane domain immediately adjacent to the signaldomain, followed by a stem domain that is immediately adjacent to thetransmembrane domain, followed by an active domain that extends to thecarboxy-terminus of the polypeptide and is located immediately adjacentto the stem domain.

Therefore, in one embodiment, an isolated nucleic acid of the inventionmay encode a truncated mammalian ST6GalNAcI polypeptide, wherein thetruncated ST6GalNAcI polypeptide is lacking all or a portion of theST6GalNAcI signal domain. In another embodiment, an isolated nucleicacid of the invention may encode a truncated mammalian ST6GalNAcIpolypeptide, wherein the truncated ST6GalNAcI polypeptide is lacking theST6GalNAcI signal domain and all or a portion of the ST6GalNAcItransmembrane domain. In yet another embodiment, a nucleic acid of theinvention may encode a truncated mammalian ST6GalNAcI polypeptide,wherein the truncated ST6GalNAcI polypeptide is lacking the ST6GalNAcIsignal domain, the ST6GalNAcI transmembrane domain and all or a portionthe ST6GalNAcI stem domain

When armed with the disclosure of the present invention, the skilledartisan will know how to make and use these and other such truncationmutants of ST6GalNAcI. In particular, when armed with the disclosure ofthe present invention, the skilled artisan will know how to make and useisolated nucleic acids encoding truncation mutants of human ST6GalNAcI,mouse ST6GalNAcI and chicken ST6GalNAcI.

The “biological activity of ST6GalNAcI” is the ability to transfer asialic acid moiety from a sialic acid donor to an acceptor molecule.Full-length human ST6GalNAcI, for example, the sequence of which is setforth in SEQ ID NO: 1, possesses such activity. The “biological activityof a ST6GalNAcI truncated polypeptide” is similarly the ability totransfer a sialic acid moiety from a sialic acid donor to an acceptormolecule. That is, a truncated ST6GalNAcI polypeptide of the presentinvention can catalyze the same glycosyltransfer reaction as thefull-length ST6GalNAcI. By way of a non-limiting example, a truncatedhuman ST6GalNAcI polypeptide encoded by an ST6GalNAcI nucleic acid ofthe invention has the ability to transfer a sialic acid moiety from aCMP-sialic acid donor to a bovine submaxillary mucin acceptor, whereinsuch a transfer results in the covalent coupling of a sialic acid moietyto a GalNAc residue on the bovine submaxillary mucin acceptor.

Therefore, a nucleic acid encoding a smaller than full-length, or“truncated,” ST6GalNAcI is included in the present invention providedthat the truncated ST6GalNAcI has ST6GalNAcI biological activity.

The methods and compositions of the invention should not be construed tobe limited solely to a nucleic acid comprising a ST6GalNAcI truncationmutant as disclosed herein, but rather, should be construed to encompassany nucleic acid encoding a ST6GalNAcI truncated mutant, prepared inaccordance with the disclosure herein, either known or unknown, which iscapable of catalyzing transfer of a sialic acid to a sialic acidacceptor. Modified nucleic acid sequences, i.e. nucleic acid sequenceshaving sequences that differ from the nucleic acid sequences encodingthe naturally-occurring proteins, are also encompassed by methods andcompositions of the invention, so long as the modified nucleic acidstill encodes a truncated protein having the biological activity ofcatalyzing the transfer of a sialic acid to a sialic acid acceptor, forexample. These modified nucleic acid sequences include modificationscaused by point mutations, modifications due to the degeneracy of thegenetic code or naturally occurring allelic variants, and furthermodifications that have been introduced by genetic engineering, i.e., bythe hand of man. Thus, the term nucleic acid also specifically includesnucleic acids composed of bases other than the five biologicallyoccurring bases (adenine, guanine, thymine, cytosine and uracil).

The present invention features an isolated nucleic acid comprising anucleic acid sequence that is at least about 90%; 95%, 97%, 98%, or 99%identical to a nucleic acid sequence set forth in any one of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 17, Δ51, SEQ ID NO:21, SEQID NO:23, Δ200, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 and SEQ IDNO:33. The present invention also features an isolated nucleic acidsequence comprising any one of the sequences set forth in SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, Δ51, SEQ ID NO:21, SEQ IDNO:23, Δ200, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33,wherein the isolated nucleic acid encodes a truncated ST6GalNAcIpolypeptide. The invention further includes an nucleic acid that encodesa truncated ST6GalNAcI polypeptide listed in Table 20.

The present invention also encompasses isolated nucleic acid moleculesencoding a truncated ST6GalNAcI polypeptide that contains changes inamino acid residues that are not essential for activity. Suchpolypeptides encoded by an isolated nucleic acid of the invention differin amino acid sequence from any one of the sequences set forth in SEQ IDNO:10, SEQ ID NO: 12, SEQ ID NO: 14, Δ35 of the human sequence shown inFIG. 31, Δ72 of the human sequence shown in FIG. 31, Δ109 of the humansequence shown in FIG. 31, Δ133 of the human sequence shown in FIG. 31,Δ170 of the human sequence shown in FIG. 31, Δ232 of the human sequenceshown in FIG. 31, Δ272 of the human sequence shown in FIG. 31, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, Δ225 of the chicken sequence shown inFIG. 31, SEQ ID NO: 18, Δ30 of the mouse sequence shown in FIG. 31, Δ51of the mouse sequence shown in FIG. 31, SEQ ID NO:22, SEQ ID NO:24 andΔ200 of the mouse sequence shown in FIG. 31; yet retain the biologicalactivity of ST6GalNAcI. By way of a non-limiting example, an isolatednucleic acid of the invention may include a nucleotide sequence encodinga polypeptide having an amino acid sequence that is at least about 90%,95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:10. Further, by way of another non-limiting example, an isolated nucleicacid of the invention includes a nucleotide sequence encoding apolypeptide that has an amino acid sequence at least about 90%, 95%,97%, 98%, or 99% identical to an amino acid sequence set forth in anyone of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, Δ35 of the humansequence shown in FIG. 31, Δ72 of the human sequence shown in FIG. 31,Δ109 of the human sequence shown in FIG. 31, Δ133 of the human sequenceshown in FIG. 31, Δ170 of the human sequence shown in FIG. 31, Δ232 ofthe human sequence shown in FIG. 31, Δ272 of the human sequence shown inFIG. 31, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, Δ225 of the chickensequence shown in FIG. 31, SEQ ID NO: 18, Δ30 of the mouse sequenceshown in FIG. 31, Δ51 of the mouse sequence shown in FIG. 31, SEQ IDNO:22, SEQ ID NO:24 and Δ200 of the mouse sequence shown in FIG. 31.

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward 1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See, generally, the internet website forthe National Center for Biotechnology Information, which is maintainedby the National Library of Medicine and the National Institutes ofHealth.

In another aspect, a nucleic acid useful in the methods and compositionsof the present invention and encoding a truncated ST6GalNAcI polypeptidemay have at least one nucleotide inserted into the nucleic acid sequenceof such a truncated mutant. Alternatively, an additional nucleic acidencoding a truncated ST6GalNAcI polypeptide may have at least onenucleotide deleted from the nucleic acid sequence. Further, a ST6GalNAcInucleic acid encoding a truncated mutant and useful in the invention mayhave both a nucleotide insertion and a nucleotide deletion present in asingle nucleic acid sequence encoding the truncated polypeptide.

Techniques for introducing changes in nucleotide sequences that aredesigned to alter the functional properties of the encoded proteins orpolypeptides are well known in the art. Such modifications include thedeletion, insertion, or substitution of bases, and thus, changes in theamino acid sequence. As is known to one of skill in the art, nucleicacid insertions and/or deletions may be designed into the gene fornumerous reasons, including, but not limited to modification of nucleicacid stability, modification of nucleic acid expression levels,modification of expressed polypeptide stability or half-life,modification of expressed polypeptide activity, modification ofexpressed polypeptide properties and characteristics, and changes inglycosylation pattern. All such modifications to the nucleotidesequences encoding such proteins are encompassed by the presentinvention.

It is not intended that methods and compositions of the presentinvention be limited by the nature of the nucleic acid employed. Thetarget nucleic acid encompassed by methods and compositions of theinvention may be native or synthesized nucleic acid. The nucleic acidmay be DNA or RNA and may exist in a double-stranded, single-stranded orpartially double-stranded form. Furthermore, the nucleic acid may befound as part of a virus or other macromolecule. See, e.g., Fasbender etal., 1996, J. Biol. Chem. 272:6479-89.

II. Vectors and Expression Systems

In other related aspects, the invention includes an isolated nucleicacid encoding a truncated ST6GalNAcI polypeptide operably linked to anucleic acid comprising a promoter/regulatory sequence such that thenucleic acid is preferably capable of directing expression of thepolypeptide encoded by the nucleic acid. Thus, the invention encompassesexpression vectors and methods for the introduction of exogenous DNAinto cells with concomitant expression of the exogenous DNA in thosecells, as described, for example, in Sambrook et al. (Third Edition,2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York).

Expression of a truncated ST6GalNAcI polypeptide in a cell may beaccomplished by generating a plasmid, viral, or other type of vectorcomprising a nucleic acid encoding the appropriate nucleic acid, whereinthe nucleic acid is operably linked to a promoter/regulatory sequencewhich serves to drive expression of the encoded polypeptide, with orwithout tag, in cells in which the vector is introduced. In addition,promoters which are well known in the art which are induced in responseto inducing agents such as metals, glucocorticoids, and the like, arealso contemplated in the invention. Thus, it will be appreciated thatthe invention includes the use of any promoter/regulatory sequence,which is either known or unknown, and which is capable of drivingexpression of the truncated ST6GalNAcI polypeptide operably linkedthereto.

In an expression system useful in the present invention, a nucleic acidencoding a truncated ST6GalNAcI polypeptide may be fused to one or moreadditional nucleic acids encoding a functional polypeptide. By way of anon-limiting example, an affinity tag coding sequence may be insertedinto a nucleic acid vector adjacent to, upstream from, or downstreamfrom a truncated ST6GalNAcI polypeptide coding sequence. As will beunderstood by one of skill in the art, an affinity tag will typically beinserted into a multiple cloning site in frame with the truncatedST6GalNAcI polypeptide. One of skill in the art will also understandthat an affinity tag coding sequence can be used to produce arecombinant fusion protein by concomitantly expressing the affinity tagand truncated ST6GalNAcI polypeptide. The expressed fusion protein canthen be isolated, purified, or identified by means of the affinity tag.

Affinity tags useful in the present invention include, but are notlimited to, a maltose binding protein, a histidine tag, a Factor IX tag,a glutathione-S-transferase tag, a FLAG-tag, and a starch binding domaintag. Other tags are well known in the art, and the use of such tags inthe present invention would be readily understood by the skilledartisan.

As would be understood by one of skill in the art, a vector comprising atruncated ST6GalNAcI polypeptide of the present invention may be used toexpress the truncated polypeptide as either a non-fusion or as a fusionprotein. Selection of any particular plasmid vector or other DNA vectoris not a limiting factor in this invention and a wide plethora ofvectors are well-known in the art. Further, it is well within the skillof the artisan to choose particular promoter/regulatory sequences andoperably link those promoter/regulatory sequences to a DNA sequenceencoding either a truncated ST6GalNAcI polypeptide. Such technology iswell known in the art and is described, for example, in Sambrook et al.(Third Edition, 2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York). Byway of a non-limiting example, a vector useful in one embodiment of thepresent invention is based on the pcWori+ vector (Muchmore et. al.,1987, Meth. Enzymol. 177:44-73).

The invention thus includes a vector comprising an isolated nucleic acidencoding a truncated ST6GalNAcI polypeptide. The incorporation of anucleic acid into a vector and the choice of vectors is well-known inthe art as described in, for example, Sambrook et al. (Third Edition,2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York).

In an aspect of the invention, an isolated nucleic acid encoding atruncated ST6GalNAcI polypeptide is integrated into the genome of a hostcell in conjunction with a nucleic acid encoding a truncated ST6GalNAcIpolypeptide. In another aspect of the invention, a cell is transientlytransfected with an isolated nucleic acid encoding a truncatedST6GalNAcI polypeptide. In another aspect of the invention, a cell isstably transfected with an isolated nucleic acid encoding a truncatedST6GalNAcI polypeptide.

For the purpose of inserting an isolated nucleic acid into a cell, oneof skill in the art would also understand that the methods available andthe methods required to introduce an isolated nucleic acid of theinvention into a host cell vary and depend upon the choice of host cell.Suitable methods of introducing an isolated nucleic acid into a hostcell are well-known in the art. Other suitable methods for transformingor transfecting host cells may include, but are not limited to, thosefound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 3rded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001), and other such laboratory manuals.

A nucleic acid encoding a truncated ST6GalNAcI polypeptide may bepurified by any suitable means, as are well known in the art. Forexample, the nucleic acids can be purified by reverse phase or ionexchange HPLC, size exclusion chromatography or gel electrophoresis. Ofcourse, the skilled artisan will recognize that the method ofpurification will depend in part on the size of the DNA to be purified.

The present invention also features a recombinant bacterial host cellcomprising, inter alia, a nucleic acid vector as described elsewhereherein. In one aspect, the recombinant cell is transformed with a vectorof the present invention. The transformed vector need not be integratedinto the cell genome nor does it need to be expressed in the cell.However, the transformed vector will be capable of being expressed inthe cell. In one aspect of the invention, a B. subtilis cell is used fortransformation of a vector of the present invention and expression ofprotein therefrom. In another aspect of the invention, E. coli is usedfor transformation of a vector of the present invention and expressionof protein therefrom. In another aspect of the invention, a K-12 strainof E. coli is useful for expression of protein from a vector of thepresent invention. Strains of E. coli useful in the present inventioninclude, but are not limited to, JM83, JM101, JM103, JM109, W3110,chi1776, and JA221.

It will be understood that a host cell useful in the present inventionwill be capable of growth and culture on a small scale, medium scale, ora large scale. For example, a host cell of the invention is useful fortesting the expression of a protein from a vector of the inventionequally as much as it is useful for large scale production of a reagentor therapeutic protein product. Techniques useful in culturing hostcells and expressing protein from a vector contained therein are wellknown in the art and will therefore not be listed herein.

A host cell useful in methods of the present invention, as describedabove, may be prepared according to various methods, as would beunderstood by the skilled artisan when amend with the disclosure setforth herein. In one aspect, a host cell of the present invention may betransformed with a vector of the present invention to produce atransformed host cell of the invention. Transformation, as known to theskilled artisan, includes the process of inserting a nucleic acid vectorinto a host cell, such that the host cell containing the nucleic acidvector remains viable. Such transformation of nucleic acid into abacterial cell is useful for purposes including, but not limited to,creation of a stably-transformed host cell, making a biological deposit,propagating the vector-containing host cell, propagating thevector-containing host cell for the production and isolation ofadditional vector, expression of target protein encoded by vector, andthe like.

Methods of transforming a cell with a vector are numerous and well-knownin the art, and will therefore not be listed here. By way of anon-limiting example, a competent bacterial cell of the invention may betransformed by a vector of the invention using electroporation. Methodsof making bacterial cells “competent” are well-known in the art, andtypically involve preparation of the bacterial cells so that the cellstake up exogenous DNA. Similarly, methods of electroporation are knownin the art, and detailed descriptions of such methods may be found, forexample, in Sambrook et al. (1989, supra). The transformation of acompetent cell with vector DNA may be also accomplished usingchemical-based methods. One example of a well-known chemical-basedmethod of bacterial transformation is described by Inoue, et al. (1990,Gene 96:23-28). Other methods of transformation will be known to theskilled artisan.

A transformed host cell of the present invention may be used to expressa truncated ST6GalNAcI polypeptide of the present invention. In anembodiment of the invention, a transformed host cell contains a vectorof the invention, which contains therein a nucleic acid sequenceencoding an truncated polypeptide of the invention. The truncatedpolypeptide is expressed using any expression method known in the art(for example, IPTG). The expressed truncated polypeptide may becontained within the host cell, or it may be secreted from the host cellinto the growth medium.

Methods for isolating an expressed polypeptide are well-known in theart, and the skilled artisan will know how to determine the best methodfor isolation of an expressed polypeptide based on the characteristicsof any given host cell expression system. By way of a non-limitingexample, an expressed polypeptide that is secreted from a host cell maybe isolated from the growth medium. Isolation of a polypeptide from agrowth medium may include removal of bacterial cells and cellulardebris. By way of another non-limiting example, an expressed polypeptidethat is contained within a host cell may be isolated from the host cell.Isolation of such an “intracellular” expressed polypeptide may includedisruption of the host cell and removal of cellular debris from theresultant mixture. These methods are not intended to be exclusiverepresentations of the present invention, but rather, are merely for thepurposes of illustration of various applications of the presentinvention.

Purification of a truncated polypeptide expressed in accordance with thepresent invention may be effected by any means known in the art. Theskilled artisan will know how to determine the best method for thepurification of a polypeptide expressed in accordance with the presentinvention. A purification method will be chosen by the skilled artisanbased on factors such as, but not limited to, the expression host, thecontents of the crude extract of the polypeptide, the size of thepolypeptide, the properties of the polypeptide, the desired end productof the polypeptide purification process, and the subsequent use of theend product of the polypeptide purification process

In an embodiment of the invention, isolation or purification of atruncated polypeptide expressed in accordance with the present inventionmay not be desired. In an aspect of the present invention, an expressedpolypeptide may be stored or transported inside the bacterial host cellin which the polypeptide was expressed. In another aspect of theinvention, an expressed polypeptide may be used in a crude lysate form,which is produced by lysis of a host cell in which the polypeptide wasexpressed. In yet another embodiment of the invention, an expressedpolypeptide may be partially isolated or partially purified according toany of the methods set forth or described herein. The skilled artisanwill know when it is not desirable to isolate or purify a polypeptide ofthe invention, and will be familiar with the techniques available forthe use and preparation of such polypeptides.

When armed with the disclosure set forth herein, the skilled artisanwould also know how to prepare a eukaryotic host cell of the invention.As set forth elsewhere herein, and as would be known to one of skill inthe art based on the disclosure provided herein, an isolated nucleicacid encoding a truncated ST6GalNAcI polypeptide may be introduced intoa eukaryotic host cell, for example, using a lentivirus-based genomicintegration or plasmid-based transfection (Sambrook et al., ThirdEdition, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (2001)). In one embodiment of the invention, aeukaryotic host cell is a fungal cell. Fungal cells useful as eukaryotichost cells of the invention include, but should not be limited to,strains such as A. niger and P. lucknowensa.

In another embodiment, a nucleic acid encoding a truncated polypeptideof the invention is cloned into a lentiviral vector containing aspecific promoter sequence for expression of the truncated polypeptide.The truncated polypeptide-containing lentiviral vector is then used totransfect a host cell for expression of the truncated polypeptide.Methods of making and using lentiviral vectors, such as those useful inthe present invention, are well-known in the art and are not describedfurther herein.

In yet another embodiment, a nucleic acid encoding a truncatedpolypeptide of the invention is introduced into a host cell using aviral expression system. Viral expression systems are well-known in theart, and will not be described in detail herein. In one aspect of theinvention, a viral expression system is a mammalian viral expressionsystem. In another aspect of the invention, a viral expression system isa baculovirus expression system. Such viral expression systems aretypically commercially available from numerous vendors.

The skilled artisan will know how to use a host cell-vector expressionsystem for the expression of a truncated polypeptide of the invention.Appropriate cloning and expression vectors for use with eukaryotic hostsarc described by Sambrook, et al., in Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor, N.Y. (2001), the disclosureof which is hereby incorporated in its entirety by reference.

Insect cells can also be used for expression of a truncated polypeptideof the present invention. In an aspect of the invention, Sf9, Sf9⁺,Sf21, High Five™ or Drosophila Schneider S2 cells can be used. In yetanother aspect of the invention, a baculovirus, or a baculovirus/insectcell expression system can be used to express a truncated polypeptide ofthe invention using a pAcGP67, pFastBac, pMelBac, or pIZ vector and apolyhedrin, p10, or OpIE3 actin promoter. In another aspect of theinvention, a Drosophila expression system can be used with a pMT or pAC5vector and an MT or Ac5 promoter.

A truncated ST6GalNAcI polypeptide of the invention of the invention canalso be expressed in mammalian cells. In an aspect of the invention,294, HeLa, HEK, NSO, Chinese hamster ovary (CHO), Jurkat, or COS cellscan be used to express a truncated polypeptide of the invention. In thecase of a mammalian cell expression of a truncated polypeptide, asuitable vector such as pT-Rex, pSecTag2, pBudCE4.1, or pcDNA/His Maxvector can be used, along with, for example, a CMV promoter. As will beunderstood by the skilled artisan, the choice of promoter, as well asmethods and strategies for introducing one or more promoters into a hostcell used for expressing a truncated ST6GalNAcI polypeptide of theinvention are well-known in the art, and will vary depending upon thehost cell and expression system used.

Various mammalian cell culture systems can be employed to expressrecombinant protein. Non-limiting examples of mammalian expressionsystems include the COS-7 lines of monkey kidney fibroblasts, describedby Gluzman, Cell 23:175 (1981), and other cell lines capable ofexpressing a compatible vector, for example, the C127, 3T3, CHO, HeLaand BHK cell tines. Mammalian expression vectors may comprise an originof replication, a suitable promoter and also any necessary ribosomebinding sites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

The methods available and the methods required to introduce any isolatednucleic acid of the invention into a host cell vary and depend upon thechoice of the host cell, as would be understood by one of skill in theart. Suitable methods of introducing an isolated nucleic acid into ahost cell are well-known in the art. By way of a non-limiting example,vector DNA can be introduced into a eukaryotic cell using conventionaltransfection techniques. As used herein, the term “transfection” refersto a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including,DEAE-dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transforming or transfecting host cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 3nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001), and other such laboratory manuals.

For example, for stable transfection of mammalian cells, it is knownthat, depending upon the expression vector and transfection techniqueused, only a small fraction of cells may integrate the foreign DNA intotheir genome. In order to identify and select these transformants, agene that encodes a selectable marker (e.g., resistance to antibiotics)is generally introduced into the host cells along with the gene ofinterest. Various selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a truncated polypeptide of theinvention or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

III. Polypeptides

A truncated polypeptide of the present invention may be truncated invarious ways, as would be known and understood by the skilled artisan,when armed with the disclosure set forth herein. Examples of truncatedpolypeptides of the present invention include, but are not limited to, apolypeptide lacking a single N-terminal residue, a polypeptide lacking asingle C-terminal residue, a polypeptide lacking both an singleN-terminal residue and a single C-terminal residue, a polypeptidelacking a contiguous sequence of residues from the N-terminus, apolypeptide lacking a contiguous sequence of residues from theC-terminus, and any such combinations thereof.

As would be understood by the skilled artisan, a lull-length humanST6GalNAcI polypeptide may contain one or more identifyable polypeptidedomains in addition to the “active domain,” the domain primarilyresponsible for the catalytic activity, of ST6GalNAcI. This is becauseit is known in that art that a full-length ST6GalNAcI polypeptidecontains a signal domain, a transmembrane domain, and a stem domain, inaddition to an active domain. Accordingly, a full-length ST6GalNAcI mayhave a signal domain at the amino-terminus of the polypeptide, followedby a transmembrane domain immediately adjacent to the signal domain,followed by a stem domain that is immediately adjacent to thetransmembrane domain, followed by an active domain that extends to thecarboxy-terminus of the polypeptide and is located immediately adjacentto the stem domain.

Therefore, in one embodiment, a ST6GalNAcI polypeptide of the inventionis a truncated mammalian ST6GalNAcI polypeptide lacking all or a portionof the ST6GalNAcI signal domain. In another embodiment, a ST6GalNAcIpolypeptide of the invention is a truncated mammalian ST6GalNAcIpolypeptide lacking the ST6GalNAcI signal domain and all or a portion ofthe ST6GalNAcI transmembrane domain. In yet another embodiment, aST6GalNAcI polypeptide of the invention is a truncated mammalianST6GalNAcI polypeptide lacking the ST6GalNAcI signal domain, theST6GalNAcI transmembrane domain and all or a portion the ST6GalNAcI stemdomain. When armed with the disclosure of the present invention, theskilled artisan will know how to make and use these and other suchtruncation mutants of human ST6GalNAcI.

The size and identity of a truncated ST6GalNAcI mutant of the presentinvention is based on the point at which the full-length polypeptide istruncated. By way of a non-limiting example, a “Δ35 human truncatedST6GalNAcI” mutant of the invention refers to a truncated ST6GalNAcIpolypeptide of the invention in which amino acids 1 through 35, countingfrom the N-terminus of the full-length polypeptide, are deleted from thepolypeptide. Therefore, the N-terminus of the Δ35 human truncatedST6GalNAcI mutant begins with the amino acid residue that would bereferred to as “amino acid 36” of the full-length polypeptide. Thisnomenclature applies to all truncated ST6GalNAcI polypeptides of theinvention, including, but not limited to those derived from mammalianST6GalNAcI, human ST6GalNAcI, mouse ST6GalNAcI and chicken ST6GalNAcIWhere specific deletions are indicated, the deletions are determinedusing the full length ST6GalNAcI sequence from chicken, mouse, or humanshown in FIG. 31. Preferred embodiments of such deletions are shown,e.g., in Table 20. In some embodiments, the truncated ST6GalNAcI mutantis selected from the following. For human truncated ST6GalNAcI mutants(using the two possible names for a single mutant): Δ35 or K36, Δ124 orK125, Δ257 or S258, Δ72 or T73, Δ109 or E110, Δ133 or M134, Δ170 orT171, Δ232 or Δ233 and Δ272 or G273. For chicken truncated ST6GalNAcImutants (using the two possible names for a single mutant): Δ48 or Q49,Δ152 or V153, Δ225 or L226, Δ226 or R227, Δ231 or K233 and Δ232 or T233.For mouse truncated ST6GalNAcI mutants (using the two possible names fora single mutant): Δ30 or K31, Δ31 or D32, Δ51 or E52, Δ126 or S127, Δ185or S186, and Δ200 or S201.

The present invention therefore also includes an isolated polypeptidecomprising a truncated ST6GalNAcI polypeptide. Preferably, an isolatedtruncated ST6GalNAcI polypeptide of the present invention has at leastabout 90% identity to a polypeptide having the amino acid sequence ofany one of the sequences set forth in SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, Δ35 of the human sequence shown in FIG. 31, Δ72 of the humansequence shown in FIG. 31, Δ109 of the human sequence shown in FIG. 31,Δ133 of the human sequence shown in FIG. 31, Δ170 of the human sequenceshown in FIG. 31, Δ232 of the human sequence shown in FIG. 31, Δ272 ofthe human sequence shown in FIG. 31, SEQ ID NO:28, SEQ ID NO:30, SEQ-IDNO:32, Δ225 of the chicken sequence shown in FIG. 31, SEQ ID NO: 18, Δ30of the mouse sequence shown in FIG. 31, Δ51 of the mouse sequence shownin FIG. 31, SEQ ID NO:22, SEQ ID NO:24 and Δ200 of the mouse sequenceshown in FIG. 31. More preferably, the isolated polypeptide is about 95%identical, and even more preferably, about 98% identical, still morepreferably, about 99% identical, and most preferably, the isolatedpolypeptide comprising a truncated ST6GalNAcI polypeptide is identicalto the polypeptide set forth in one of SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO:14, Δ35 of the human sequence shown in FIG. 31, Δ72 of the humansequence shown in FIG. 31, Δ109 of the human sequence shown in FIG. 31,Δ133 of the human sequence shown in FIG. 31, Δ170 of the human sequenceshown in FIG. 31, Δ232 of the human sequence shown in FIG. 31, Δ272 ofthe human sequence shown in FIG. 31, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, Δ225 of the chicken sequence shown in FIG. 31, SEQ ID NO: 18, Δ30of the mouse sequence shown in FIG. 31, Δ51 of the mouse sequence shownin FIG. 31, SEQ ID NO:22, SEQ ID NO:24 and Δ200 of the mouse sequenceshown in FIG. 31.

The present invention also provides for analogs of polypeptides whichcomprise a truncated ST6GalNAcI polypeptide as disclosed herein. Analogscan differ from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

Fragments of a truncated ST6GalNAcI polypeptide of the invention areincluded in the present invention, provided the fragment possesses thebiological activity of the full-length polypeptide. That is, a truncatedST6GalNAcI polypeptide of the present invention can catalyze the sameglycosyltransfer reaction as the full-length ST6GalNAcI. By way of anon-limiting example, a truncated human ST6GalNAcI polypeptide of theinvention has the ability to transfer a sialic acid moiety from aCMP-sialic acid donor to a bovine submaxillary mucin acceptor, whereinsuch a transfer results in the covalent coupling of a sialic acid moietyto a GalNAc residue on the bovine submaxillary mucin acceptor.Therefore, a smaller than full-length, or “truncated,” ST6GalNAcI isincluded in the present invention provided that the truncated ST6GalNAcIhas ST6GalNAcI biological activity.

In another aspect of the present invention, compositions comprising anisolated truncated ST6GalNAcI polypeptide as described herein mayinclude highly purified truncated ST6GalNAcI polypeptides.Alternatively, compositions comprising truncated ST6GalNAcI polypeptidesmay include cell lysates prepared from the cells used to express theparticular truncated ST6GalNAcI polypeptides. Further, truncatedST6GalNAcI polypeptides of the present invention may be expressed in oneof any number of cells suitable for expression of polypeptides, suchcells being well-known to one of skill in the art, as described indetail elsewhere herein.

It will be appreciated that all above descriptions of a truncatedST6GalNAcI polypeptide applies equally to truncated ST6GalNAcIpolypeptides of the invention from any source, including, but notlimited to mammalian ST6GalNAcI, human ST6GalNAcI, mouse ST6GalNAcI, andchicken ST6GalNAcI.

Substantially pure protein isolated and obtained as described herein maybe purified by following known procedures for protein purification,wherein an immunological, enzymatic or other assay is used to monitorpurification at each stage in the procedure. Protein purificationmethods are well known in the art, and are described, for example inDeutscher et al. (ed., 1990, Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego). In a preferred embodiment, the truncatedST6GalNAcI polypeptides of the invention are fused to a purificationtag, e.g. a maltose binding domain (MBD) tag or a starch binding domain(SBD) tag. Such truncated ST6GalNAcI fusion proteins can be purified bypassage through a column that specifically binds to the purificationtag, e.g., MBD or SBD proteins can be purified on a cyclodextrin column.In a further embodiment, a truncated ST6GalNAcI fusion proteinscomprising a purification tag, such as, e.g., an MBD or SBD tag, areimmobilized on a column that specifically binds to the purification tagand substrates, e.g., a sialic acid donor or PEGylated-sialic acid donorand a glycoprotein or glycopeptide comprising an O-linked glycylationsite are passed through the column under conditions that facilitatetransfer of sialic acid from a donor, e.g., CMP-sialic acid orCMP-PEGylated-sialic acid, to a glycoprotein or glycopeptide acceptor,and thus production of a sialylated glycoprotein or sialylatedglycopeptide.

III. Methods

The present invention features a method of expressing a truncatedpolypeptide. Polypeptides which can be expressed according to themethods of the present invention include a truncated ST6GalNAcIpolypeptide. More preferably, polypeptides which can be expressedaccording to the methods of the present invention include, but are notlimited to, a truncated human ST6GalNAcI polypeptide, a truncated mouseST6GalNAcI polypeptide, and a truncated chicken ST6GalNAcI polypeptide.In a preferred embodiment, a polypeptide which can be expressedaccording to the methods of the present invention is a polypeptidecomprising any one of the polypeptide sequences set forth in SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, Δ35 of the human sequence shown inFIG. 31, Δ72 of the human sequence shown in FIG. 31, Δ109 of the humansequence shown in FIG. 31, Δ133 of the human sequence shown in FIG. 31,Δ170 of the human sequence shown in FIG. 31, Δ232 of the human sequenceshown in FIG. 31, Δ272 of the human sequence shown in FIG. 31, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, Δ225 of the chicken sequence shown inFIG. 31, SEQ ID NO: 18, Δ30 of the mouse sequence shown in FIG. 31, Δ51of the mouse sequence shown in FIG. 31, SEQ ID NO:22, SEQ ID NO:24 andΔ200 of the mouse sequence shown in FIG. 31.

In one embodiment, the present invention features a method of expressinga truncated ST6GalNAcI polypeptide encoded by an isolated nucleic acidof the invention, as described elsewhere herein, wherein the expressedtruncated ST6GalNAcI polypeptide has the property of catalyzing thetransfer of a sialic acid moiety to an acceptor moiety. In one aspect ofthe invention, a method of expressing a truncated ST6GalNAcI polypeptideincludes the steps of cloning an isolated nucleic acid of the inventioninto an expression vector, inserting the expression vector constructinto a host cell, and expressing a truncated ST6GalNAcI polypeptidetherefrom.

Methods of expression of polypeptides, as well as construction ofexpression systems and recombinant host cells for expression ofpolypeptides, are discussed in extensive detail elsewhere herein.Methods of expression of a truncated polypeptide of the presentinvention will be understood to include, but not to be limited to, allsuch methods as described herein. In some expression systems, thetruncated ST6GalNAcI polypeptides of the invention are expressed asinsoluble proteins, e.g., in an inclusion protein in a bacterial hostcell. Methods of refolding insoluble glycosyltransferases, includingST6GalNAcI polypeptides, are disclosed in U.S. Provisional PatentApplication Ser. No. 60/542,210, filed Feb. 4, 2004; U.S. ProvisionalPatent Application Serial No: 60/599,406, filed Aug. 6, 2004; U.S.Provisional Patent Application Ser. No. 60/627,406, filed Nov. 12, 2004;and International Patent Application No. PCT/US05/03856, filed Feb. 4,2005; each of which are herein incorporated by reference for allpurposes.

The present invention also features a method of catalyzing aglycosyltransferase reaction between a glycosyl donor and a glycosylacceptor. In one embodiment, the invention features a method catalyzingthe transfer of a sialic acid moiety to an acceptor moiety, wherein thesialyltransfer reaction is carried out by incubating a truncatedST6GalNAcI polypeptide of the invention with a sialic acid donor moietyand an acceptor moiety. In one aspect, a truncated ST6GalNAcIpolypeptide of the invention mediates the covalent linkage of a sialicacid moiety to an acceptor moiety, thereby catalyzing the transfer of asialic acid moiety to an acceptor moiety.

In an embodiment of the invention, a truncated ST6GalNAcI polypeptideuseful in a glycosyltransfer reaction is a truncated human ST6GalNAcIpolypeptide. In another embodiment, a truncated ST6GalNAcI polypeptideuseful in a glycosyltransfer reaction is a truncated chicken ST6GalNAcIpolypeptide. In a preferred embodiment, a truncated ST6GalNAcIpolypeptide useful in a glycosyltransfer reaction is a polypeptidecomprising any one of the polypeptide sequences set forth in SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, or any of the human truncated ST6GalNAcI polypeptides listed inTable 20.

By way of a non-limiting example, a method of catalyzing the transfer ofa sialic acid moiety to an acceptor moiety includes the steps ofincubating a truncated human ST6GalNAcI polypeptide with acytidinemonophosphate-sialic acid (CMP-NAN) sialic acid donor and anasialo bovine submaxillary mucin acceptor moiety, wherein the truncatedhuman ST6GalNAcI polypeptide mediates the transfer of a sialic acidmoiety from CMP-NAN to the bovine submaxillary mucin acceptor.

Therefore, in one embodiment, the present invention also features apolypeptide acceptor moiety. In one embodiment of the invention, apolypeptide acceptor moiety is a human growth hormone. In anotherembodiment, a polypeptide acceptor moiety is an erythropoietin. In yetanother embodiment, a polypeptide acceptor moiety is aninterferon-alpha. In another embodiment, a polypeptide acceptor moietyis an interferon-beta. In another embodiment of the invention, apolypeptide acceptor moiety is an interferon-gamma. In still anotherembodiment of the invention, a polypeptide acceptor moiety is alysosomal hydrolase. In another embodiment, a polypeptide acceptormoiety is a blood factor polypeptide. In still another embodiment, apolypeptide acceptor moiety is an anti-tumor necrosis factor-alpha. Inanother embodiment of the invention, a polypeptide acceptor moiety isfollicle stimulating hormone. In yet another embodiment of theinvention, a polypeptide acceptor moiety is a glucagon-like peptide.

In one embodiment, the present invention also features a method oftransferring a sialic acid-polyethyleneglycol conjugate (SA-PEG) to anacceptor molecule. In one aspect, an acceptor molecule is a polypeptide.In another aspect, an acceptor molecule is a glycopeptide. Compositionsand methods useful for designing, producing and transferring a SA-PEGconjugate to an acceptor molecule are discussed at length inInternational (PCT) Patent Application No. WO03/031464 (PCT/US02/32263)and U.S. Patent Application No 2004/0063911, each of which isincorporated herein by reference in its entirety.

Methods of assaying for glycosyltransferase activity are well-known inthe art. Various assays for detecting glycosyltransferases which can beused in accordance with the invention have been published. The followingare illustrative, but should not be considered limiting, of those assaysuseful for detecting glycosyltransferase activity. Furukawa et al (1985,Biochem. J., 227:573-582) describe a borate-impregnated paperelectrophoresis assay and a fluorescence assay Roth et al (1983, Exp'lCell Research 143:217-225) describe application of the borate assay toglucuronyl transferases, previously assayed calorimetrically. Benau etal (1990, J. Histochem. Cytochem., 38:23-30) describe a histochemicalassay based on the reduction, by NADH, of diazonium salts. See also U.S.Pat. No. 6,284,493 of Roth, incorporated herein by reference.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Example 1 Molecular Cloning of Mouse GalNAc α2,6-Sialyltransferase(ST6GalNAcI) into the MBP-pCWin2 Vector

The cloning and expression of five N-terminal amino acid truncatedGalNAc α2, 6-Sialyltransferase (ST6GalNAcI) genes into the pCWin2 MBPfusion tag expression vector was conducted as described herein. Alsodescribed herein is the generation of five different amino-terminaltruncations of the ST6GalNAcI gene fused to Maltose binding protein(MBP) in the pCWin2-MBP vector. Generation of JM109 cells transformedwith these constructs and the subsequent induction of protein expressionin these transformants is presented. All five fusion proteins areexpressed at varying levels upon induction with IPTG.

Template DNA (pTS103) was used for amplification of mouse ST6GalNAcI.Primers were designed to clone mouse ST6GalNAcI gene using the followingsequences for five N-terminal truncated forms of mouse ST6GalNAcI,including Δ31, Δ51, Δ126, Δ185 and Δ200. The primers used were asfollows:

D32-HindIII-5′-taatataagottgatccaagggcaaaagattc-3′ (SEQ ID NO:43),E52-BamHI-5′-taataaggatcogagattctgcaa aaggctga-3 (SEQ ID NO:44),S127-BamHI-5′-taatatggatcctcagaacacctggacaaa gt-3′(SEQ ID NO:45),S186-BamHI-5′-taatatggatcctctgagcctcggtgggattt-3(SEQ ID NO:46),S201-13BamHI-5′-taataaggatccagcagcctgcagacgaactg-3(SEQ ID NO-47), andM-XhoI-5′-tag cgc ctc gag tca gtt ctt tgc ttt gtc act ttg-3′(SEQ IDNO):48). A PCR reaction was conducted in autoclaved 500 μl reactiontubes for amplification of various ST6GalNAcI genes.

TABLE 1 PCR reaction parameters for mouse ST6GalNAcI truncation mutants.Reaction tubes Reagents D32 E52 S127 S186 S201 10X Herculase Buffer 5 μl5 μl 5 μl 5 μl 5 μl 25 mM MgCl2 1 μl 1 μl 1 μl 1 μl 1 μl 10 mM dNTP 1 μl1 μl 1 μl 1 μl 1 μl Forward primer 10 pmol/μl 4 μlA 4 μlB 4 μlC 4 μlD 4μlE Reverse primer 10 pmol/μl 4 μlF 4 μlF 4 μlF 4 μlF 4 μlF Nucleasefree water 31 μl 31 μl 31 μl 31 μl 31 μl Template (pTs 103) 12.4 ng 3 μl3 μl 3 μl 3 μl 3 μl Herculase polymerase 1 μl 1 μl 1 μl 1 μl 1 μl Lidtemperature 105° C.  STEP 1 92° C.  45 Seconds STEP 2 61° C.  60 secondsSTEP 3 72° C. 3.0 Minutes. STEP 1, 2 and 3 30 Cycles. STEP 4 92° C.  45Seconds STEP 5 61° C.  60 Seconds STEP 6 72° C.  10 Minutes. STEP 4, 5and 6  4 Cycles. STEP 7  4° C. PAUSE

The results of the PCR reaction were visualized using 0.8% agarose/TAEgels. The ST6GalNAcI gene was identified at about 1.5 Kb. DNA wasextracted from the get using Amicon Ultra free DA filters and purifiedusing Microcon YM-100 filters, according to manufacturer's instructions(Millipore, Bellerica, Mass.).

A DNA band around 1.5 Kb in the 0.8% agarose gel was identified using aUV transilluminator. A gel slice containing the DNA was excised from thegel. Using an Amicon Ultra free DA filter Millipore, Bellerica, Mass.),the gel slice was placed in a gel nebulizer and the device sealed withthe cap attached to the vial. The assembled device was centrifuged for10 minutes at 5000×g. The extruded DNA passed through the microporousmembrane in the sample filter cup and was collected in the filtratevial. Purified DNA in the vial was transferred into a sample reservoirof a Microcon YM-100 unit (Millipore, Bellerica, Mass.) and centrifugedat 200 rpm for 12 minutes. The transferred DNA was collected.

Restriction enzyme digestion of concentrated DNA from the PCR reactionwas conducted in a 1.5 ml tube by adding 6.0 μl of purified PCR product,2.5 μl of 10× Bam HI buffer, 2.5 μl of 10×BSA, 1.5 pt of Bam HI enzyme,1.5 μl XhoI enzyme, and 11.0 μl nuclease free water. Reactions wereincubated for 1.5 hours at 37 o C and placed on ice for 5 minutes.MBP-pCWin2 vector DNA was digested in a 1.5 ml tube by adding 6.0 μlvector DNA (MBP-pCWin2), 2.5 μl 10× Bam HI buffer, 2.5 μl 10×BSA, 1.5 μlBamHI enzyme, 1.5 μL XhoI enzyme, and 11.0 μl nuclease free water. Thedigestion reaction was analyzed by electrophoresis on 0.8% agarose/TAEgels. Gels were loaded with digestion mixtures containing 2 μl ofloading dye and 10 μl of digested DNA. DNA around 1-5 Kb was extractedfrom the gel using the Amicon Ultra free DA protocol and purified usingMicrocon YM-100 according to manufacturer's instructions (Millipore,Bellerica, Mass.).

In autoclaved 0.5 ml tubes, the following BamHI/XhoI digested DNA wasadded in order to ligate the insert into the vector:

TABLE 2 Ligation reactions for mouse ST6GalNAcI truncation mutants.HindIII/XhoI digested DNA 1 2 3 4 5 D32 11.5  — — — — E52 — 11.5  — — —S127 — — 11.5  — — S186 — — — 11.5  — S201 — — — — 11.5  μl Bam HI/XhoIdigested 1.5 1.5 1.5 1.5 1.5 MBP-pCWIn2 μl 10× ligation buffer 1.5 1.51.5 1.5 1.5 μl T4 DNA ligase 0.5 0.5 0.5 0.5 0.5

Reaction mixtures were incubated at 4° C. overnight.

To each of five pre-chilled 2 mm gap cuvettes numbered 1, 2, 3, 4 and 5was added 2.0 μl of the ligation reactions listed in Table 2. Mixtureswere added to corresponding cuvettes including 50 μl of thawed (on ice)DH5α electrocompetent cells. The mixture was subject to electroporationat 2.5 KV, R5 resistance and 129 OHMS. SOC media (1 ml) was added toeach reaction mixture, which was then incubated at 37 o C for one hourwith shaking at 225 RPM. 100 μl of each transformation reaction wasplated onto LB (50 μg/ml) Kanr plates and incubated at 37oC overnight.

For positive clone screening, four transformant colonies were selectedfrom each construct and were inoculated into 5.0 ml of LB brothcontaining 10 μg/ml of Kanamycin and grown at 37o C for 5 hours, withshaking (225 rpm). DNA was isolated using a QIA prep Spin Miniprep Kitaccording to manufacturer's instructions (Qiagen, Valencia, Calif.,Valencia, Calif.). Plasmid DNA was prepared with both BamHI/XhoI asdescribed previously. The digestion reactions then were then analyzed on0.8% agarose/TAE gels.

DNA from colonies #1 through #4, construct DH5α/MBP-pCWin2-ST6GalNAcI(D32, E52, S127, S186, S201, corresponding to Δ31, Δ51, Δ126, Δ185, andΔ200, respectively), was double digested using restriction enzymes NdeIand HindIII as set forth in Table 3 in order to isolate MBP-ST6GalNAcIfragments.

TABLE 3 Diagnostic conditions for ST6GalNAcI truncation mutant DNAisolates. In 0.5 ml autoclaved tubes: 6.0 μl DNA from each mutantDH5α/pCWin2-MBP-ST6GalNAcI 2.5 μl 10X NEB4 Buffer 2.5 μl 10X BSA 1.5 μlNdeI enzyme 1.5 μl XhoI enzyme 11.0 μl  Nuclease free water

Reactions were incubated at 37° C. for 1.5 hours. The digestionreactions were then analyzed on 0.8% agarose/TAE gels.

Five positive clones from each truncated ST6 GalNAcI (Colony #1) wereinserted into E. coli JM109 cells for expression. To five 1.5 mlautoclaved eppendorf tubes labeled D32, E52, S127, S186 and S201 wasadded 50 μl of JM109 chemically competent cells, 2.0 μl of mini-prep DNAcolony #1 (corresponding to tubes D32, E52, S127, S186 and S201) fromconstruct DH5α/MBP-pCWin2-ST6GalNAcI. The mixtures were incubated on icefor 30 minutes, then heat-shocked for 30 seconds at 42 o C withoutshaking. Immediately after heat shocking, the tubes were transferred toice. Room temperature SOC medium (250 μl) was added and the tubes wereshaken horizontally at 225 rpm at 37 o C for one hour. A volume of 150μl of each culture was spread onto LB (50 mg/ml) Kanr agar plates andincubated at 37oC overnight.

DNA from Col. #1 and Col. #2 constructs JM109/MBP-pCWin2-ST6GalNacI(D32, E52, S127, S186 and S201) was then double-digested usingrestriction enzymes NdeI and XhoI as follows in order to get theMBP-ST6GalNAcI fragment isolated. Digestion conditions are shown inTable 4.

TABLE 4 Digestion conditions for MBP-pCWin2-ST6GalNacI constructs 6.0 μlDNA from JM109/pCWin2-MBP-GnT1 2.5 μl 10X NEB4 Buffer 2.5 μl 10X BSA 1.5μl NdeI enzyme 1.5 μl XhoI enzyme 11.0 μl  Nuclease free water

Vials were incubated at 37° C. for 1.5 hours. The digestion reactionthen was analyzed on 0.8% agarose/TAE gels.

Mouse GalNAc α2,6-Sialyltransferase (ST6GalNAcI) was expressed fromJM109 cells harboring MBP-pCWin2-ST6GalNAcI. 150 ml Martone L-brothcontaining 10 μg/ml of Kanamycin was innoculated with colony #1 of eachN-terminal amino acid truncated construct of JM109/pCWin2-MBP-ST6GalNAcI(D32, E52, S127, S186, and S201). The optical density was monitored at620 nm until the culture reached an OD of 0.7. Protein expression wasinduced overnight at 35oC by addition of IPTG (final concentration=500mM). The next day, the culture was harvested by centrifugation at 4oC,5000 rpm for 30 minutes. The pellet was resuspended in distilled water.For each gram of pellet, 3.3 ml of water were added. Cells weredisrupted using a French press, and the lysed cells were centrifuged at10000 rpm for 20 minutes. Cell pellets were separated from cellsupernatant and an SDS page gel was used to visualize the samples.

SDS-PAGE was conducted using Novex pre-cast 4-20% Tris-Glycine gels inNovex XCELL Electrophoresis System (Invitrogen, Carlsbad, Calif.).Samples were prepared by mixing 50 μl of protein solution with 50 μl of2× loading buffer and 10 μl of 1M DTT followed by heating at 98 o C for4 minutes. A volume of 10 μl of each sample was loaded onto the gel andsubjected to a constant voltage of 100 V. When the marker dye reachedthe bottom of the gel, the get was washed with water 3 times for 5minutes each time. The gel was stained for one hour at room temperaturewith gentle shaking. The gel was destained with water to obtain a clearbackground.

TABLE 5 Number of colonies resulting from 100 μl of inoculum forelectroporation of E. coli DH5α. Table of all transformants ConstructsDH5α ST6GalNAcI No. of colonies D32 84 E52 372 S127 88 S186 225 S201 232

TABLE 6 Number of colonies resulting from 150 μl of inoculum forelectroporation of E. coli JM109 host cells Table of all transformantsConstructs JM109 ST6GalNAcI No. of colonies D32 6 E52 21 S127 12 S186 6S201 21

FIG. 2 illustrates the DNA obtained from PCR, after restriction digestsusing both endonucleases. Expected DNA fragments of 1488 bp, 1428 bp,1203 bp, 1026 bp, and 981 bp correspond respectively to D32, E52, S127,S186, and S201 of N-terminal amino acid truncated ST6GalNAcI. FIG. 3illustrates the screening of recombinant coloniesDH5α/pCWin2-MBP-ST6GalNAcI, wherein the DNA was digested usingHindIII/XhoI restriction enzyme for D32 product and BamHI/XhoI for theconstructs E52, S127, S186 and S201 products.

In summary, five mouse N-terminal amino acid truncated GalNAcα2,6-sialyltransferase (ST6GalNAcI) constructs have been successfullycloned and transformed into E. coli DH5α and JM109 host cells, as shown.Construct S201, representing ST6GalNAcI Δ200, was further confirmed bysequence analysis. Fusion proteins have been expressed from E. coliJM109 host cells. The E. coli JM109 transformants have been shown toexpress the correct size ST6GalNAcI-MBP fusion proteins on SDS page gel.

Example 2 Development of Protein Refolding Conditions for E. ColiExpressed MBP-Mouse ST6GalNAcI

E. coli-expressed fusion proteins of Maltose Binding Protein (MBP) and atruncated Mouse GalNac α2,6-Sialyltransferase (ST6GalNAcI) were examinedand refolded to produce an active enzyme. For this work, enzyme activityis defined as transfer of sialic acid on to an acceptor proteingranulocyte-colony stimulating factor (G-CSF)-O-GalNac by ST6GalNAcI,using a CMP-NAN donor.

Refolding experiments on MBP-ST6GalNAcI were carried out on a 1 mlscale, with five different MBP-ST6GalNAcI DNA constructs and 16different possible refolding conditions. Refolding was performed usingthe Hampton Research Foldit kit (Hampton Research, Aliso Viejo, Calif.)and the assays were performed via radioactive detection of CMP [14C]sialic acid addition to a Asialo Bovine Submaxillary Mucin (A-BSM) orAsialo Fetuin (AF), using matrix-assisted laser desorption ionizationmass spectrometry (MALDI) analysis utilizing addition of sialic acid toG-CSF-O-GalNAc. The data shows that E. coli-expressed MBP-ST6GalNAcI canbe refolded into an active enzyme. Under refold condition 8 found inHampton Research's Foldit kit (Hampton Research, Aliso Viejo, Calif.),as described herein, active conformations of MBP-ST6GalNAcI constructS201 (serine 201) were obtained. This was validated by a CMP[14C]-sialic acid ST6GalNAcI assay and later confirmed by aGalNAc−O-G-CSF assay.

Glycerol stocks of JM109 pCWin2 MBP-ST6GalNAcI constructs were prepared.Assembly of these constructs is described elsewhere herein. Theconstructs are comprised of different amino terminal amino acidtruncations from the original Mouse ST6GalNAcI protein; includingConstruct 1—pCWin2 MBP-ST6GalNAcI-D32 Aspartic acid (496aa, 57115.13MW); Construct 2—pCWin2 MBP-ST6GalNAcI-E52 Glutamic acid (476aa,54814.77 MW); Construct 3—pCWin2 MBP-ST6GalNAcI-S127 Serine (401aa,46562.77 MW); Construct 4—pCWin2 MBP-ST6GalNAcI-S186 Serine (342aa,40160.65 MW); and Construct 5—pCWin2 MBP-ST6GalNAcI-S201 Serine (327aa,38245.82 MW).

Constructs were grown in 150 ml Martone L-Broth cultures containing 10μg/ml Kanamycin sulfate. Each culture was inoculated with one isolatedcolony corresponding to constructs #1 through #5. The 150 ml cultureswere incubated overnight at 37° C., shaking at 250 rpm. Starter culturesof 5 ml Martone L-Broth containing 10 μg/ml Kanamycin sulfate wereinoculated with one isolated colony of construct S186 and S201. Thisprocedure was performed for a total of four starter cultures. Startercultures were incubated overnight at 37° C., shaking at 250 rpm.

Lastly, two 1 L Martone L-Broth cultures containing 10 μg/ml Kanamycinsulfate were prepared. Each of these cultures was inoculated with 5 mlof over night starter culture of constructs S186 or S201. These 1 Lcultures were incubated at 37° C., with shaking at 250 rpm, until theOD620 measured in a range of 0.6 to 1.0. Upon reaching this point, IPTGwas added to each of the two 1 L cultures to a final concentration of0.5 mM. Cultures were then allowed to continue incubating overnight at37° C., with shaking at 250 rpm. In addition, two fermenter vesselscontaining 1½ liter of Martone L-Broth with 10 μg/ml Kanamycin wasinoculated with 5.0 ml of starter culture with following unitspecifications: temperature 37.0, pH=7.0.

Cultures (150 ml) of JM109 pCWin2 MBP-ST6GalNAcI constructs 1 through 5were transferred to 250 ml centrifuge bottles. Cultures were thencentrifuged at 5000 rpm for 30 minutes at 4° C. Supernatants wereremoved and the pellets were weighed. The pellets from each sample werethen washed to isolate the inclusion bodies (IBs). The pellet of eachconstruct was first resuspended in 6.0 ml of 20 mM Tris-HCl, 5 mM EDTA,pH-9 and then lysed by adding 25 μl of 20 mg/ml lysozyme and 10 μl of 1mg/ml DNase1. The reaction tubes then were incubated at 37oC for onehour.

The lysates for each construct were then centrifuged at 10,000 rpm at,4oC for 15 minutes. The supernatants were removed and the pellets wereresuspended in 6.0 ml of 20 mM TrisHcl, 5 mM EDTA, pH=6.5. Thesupernatants were then removed and the pellets were resuspended a secondtime in 6.0 ml of 20 mM Tris-Hcl, 5 mM EDTA pH=6.5. The suspensions werethen centrifuged at 5000 rpm, 25° C. for 5 minutes. The supernatantswere removed and a third wash was performed by resuspending the pelletsin 6.0 ml of 20 mM Tris-HCl, pH=6.5, 5 mM EDTA. The suspensions werethen centrifuged at 5000 rpm, 25° C. for 5 minutes. The supernatantsfrom each sample were removed and the pellets were weighed and stored at−20oC. SDS-PAGE was conducted using both the lysates and the pellets byadding 50 μl of the sample and 50 μl of 2× loading buffer and 10 μl of1.0 M DTT heating at 98oC for 6 minutes. Expression of the protein wasobserved in the gel. The pellets were then weighed and resuspended with1.0 ml of 20 mM Tris-HCl pH=6.5, 5 mM EDTA. 1 ml aliquots were made foreach of the five constructs and used for analysis. These aliquotsrepresent the triple washed inclusion bodies (TWIsB).

Cultures from JM109/pCWin2-MBP-ST6GalNAcI constructs S186 and S201 inshaker flasks and fermenters were transferred to 1 L centrifuge bottles.Cultures were then centrifuged at 5000 rpm for 30 minutes at 4oC.Supernatants were removed and the pellets were weighed. The pellets fromeach sample were then washed to isolate the inclusion bodies (IB's). Thepellets of S186 and S201 were first resuspended in 35 ml of 20 mMTris-HCl, PH=8-0, 5 mM EDTA and then lysed by two passages through theFrench press at 12,000 psi.

The lysates for each construct were then centrifuged at 5000 rpm, 25oCfor 5 minutes in 50 ml disposable tubes. The supernatants were removedand the pellets were resuspended in 35 ml of 20 mM Tris HCl, pH=6.5, 5mM EDTA. The suspensions were then centrifuged and the samples wereresuspended a second time in 35 ml of 20 mM Tris-HCl, pH-6.5, 1% TritonX-100. The suspensions were again centrifuged at 5000, 25oC for 5minutes. The supernatants were removed and a third wash was performed byresuspending the pellets in 35 ml of 20 mM tris-HCl pH=6.5, 5 mM EDTA.The suspensions were then centrifuged at 5000 rpm, 25° C. for 5 minutes.The supernatants from each sample were removed and the pellets wereweighed and stored at −20oC.

Solubilization buffer was prepared with the following concentrations ofmaterials: 6M Guanidine HCl, 5 mM EDTA, 50 mM Tris-HCl, pH=6.5 and 10 mMDTT. 1 ml of this solution was added to 20 mg TWIBs to yield a 20 mg/mlsolution. The solution was incubated overnight on the bench top tosolubilize IBs. This procedure was performed on a TWIB aliquot of eachMBP-ST6GalNAcI construct to provide protein for refolding experiments.Protein samples from each construct were diluted by combining 950 μl ofIB solubilized butter with 50 μl of protein sample. Samples were thenanalyzed by UV Spectrophotometer and the protein concentration andpercent protein solubilized conversion was calculated from those valuesand the molar extinction coefficient: Construct D32-1.24 mg/ml per 1A280 unit, Construct E52-1.29 mg/ml per 1 A280 unit, Construct S127-1.52mg/ml per 1 A280 unit, Construct S186-1.77 mg/ml per 1 A280 unit,construct S201-1.38 mg/ml per 1 A280 unit.

Protein refold samples were purified using Harvard Bioscience G-50 MacroSpin Columns (Holliston, Mass.). Caps were removed from the G-50 columnsand these were placed into 2 ml microcentrifuge tubes. 500 μl of waterwas added to each column and they were then allowed to incubate for 15minutes to hydrate. The columns were then centrifuged at ˜2000×g for 4minutes after which they were transferred to new 2 ml centrifuge tubes.1501 of each refold solution was applied to one of the columns. Columnswere then centrifuged at 2000×g for 2 minutes. Resulting permeatesrepresented the purified refold samples. An SDS gel was used tovisualize the purified protein

To screen refolding conditions that may result in an active form of Ecoli expressed MBP-ST6GalNAcI, a Hampton Foldit Screening kit (HamptonResearch, Aliso Viejo, Calif.) was utilized. The composition of each ofthe refolding buffers is set forth elsewhere herein. For a givenrefolding condition, 950 μl of refolding buffer was combined with 50 μlof solubilized protein (for high protein concentration conditions) or950 μl of refolding buffer was combined with 50 μl of 1:10 dilution ofthe high protein concentration of solubilized protein (for low proteinconcentration conditions). Refolding reactions were placed on a rotatorin a cold room (4° C.), rotating overnight.

A radiolabeled [14C] CMP-sialic acid assay was performed to determinethe activity of the E. coli expressed refolded MBP-ST6GalNAcI bymonitoring the addition of radiolabel to Asialo Fetuin (AF) or A-BSM(Asialo Bovine Submaxillary glands Mucin) acceptor. 50 mg of AF wasdissolved in 11.0 ml of water to have an initial concentration of 50mg/ml. A-BSM was prepared by release of sialic acid by means ofhydrolysis from BSM (mucin, type 1-S). The initial screen was performedon refolded protein samples obtained in 150 ml cultures. Subsequentrefold samples were also refolded and purified from one liter culturesfor construct S201 and S186. The assay included protein samples,ST6GalNAcI from baculovirus as a positive control, a negative controlsample with all the components except acceptor and a maximum inputsample which contained all components except enzyme. A total of 20samples were tested. The 14C ST6GalNAcI assay reaction mixture included50 mg/ml A-BSM or AF at 0.25 mg, in 50 mM MES pH 6.0, 100 mM NaCl 40 nCi[14C]-CMP-sialic acid, 0.2 mM cold CMP sialic acid, with 10 μl enzymesolution.

For each of the refolding samples, 40 μl of the reaction mixture werecombined with 10 μl of the refolding samples. For the negative control10 μl H2O was combined with 40 μl of the reaction mixture. Positivecontrol was treated the same as samples that is addition of 10 μl ofST6GalNAcI baculovirus enzyme supernatant was added to 40 μl reactionmixture. For the maximum input sample 40 μl of the reaction mixture wascombined with 10 μl of dH2O. Reactions were incubated at 37° C. for 60minutes. Reactions were stopped by addition of 100 μl of mixture of 5%phosphotungstic acid/15% TCA. The reaction mixture was microfuged at10000 rpm for two minutes. Supernatant was removed by pipetting and thesediments were washed with 500 μl of 5% TCA and vortexed. The mixturewas microfuged at 10,000 rpm for two minutes and the supernatant wasremoved by pipetting. The pellets were resuspended in 100 μl of 10NNaOH. One-ml of H2O was added to each reaction; samples were vortexedbriefly and then loaded into scintillation vials. Five-ml ofscintillation cocktail was added to each of the samples and controlsSamples were shaken briefly and loaded on the scintillation counter andradioactivity measured.

A G-CSF assay was performed to determine whether E. coli-expressedrefolded MBP-ST6GalNAcI, in the presence of CMP-NAN, could transfersialic acid to a GalNAc−O-G-CSF acceptor. ST6GalNAcI construct S 186(refold buffers #8 and #11) and construct S201 (refold buffer # 8) wereassayed for transferase activity. Additionally, as a positive control,ST6GalNAcI from Baculovirus was assayed. The assay includedGalNAc−O-GCSF (100 μg), CMP-NAN (0.750 mg), MES buffer, pH 6.0, andMnCl2 (100 mM). Table 7 illustrates the silayltransferase reaction ascataylzed by the enzyme obtained by refold condition #8.

TABLE 7 Sialyltransferase activity of enzyme obtain under refoldingcondition #8. Transfer of Sialic acid by Bacterial ST6GalNAcI refold #8to GalNac-G-CSF. Reaction mixture A B 1-GalNAc G-CSF 1 μg/μl  50 μl  50μl 2-MnCl2 100 mM 5.0 μl  5.0 μl 3-CMP-NAN 5.0 μl  5.0 μl ST6GalNAc I 50 μl 100 μl GalNAc-G-CSF dissolved in 25 mM of MES Buffer + 0.05% ofNa azide pH = 6.0. CMP-NAN 0.75 g in 100 μl of MES Buffer. ST6GalNAc IRefold #8. Incubate reaction tubes at 32° C. with gentle shaking. Takeout 5.0 μl each time and submit for MALDI-TOF analysis.

At different time intervals (2, 24, 48, and 120 hrs), aliquots ofsamples were subjected to MALDI-time of flight (TOF) analysis. Resultsclearly indicate transfer of sialic acid to GalNac-O-G-CSF.

Pellet weights and inclusion body weights were determined for each ofthe five 150 ml JM109 pCWin MBP-ST6GalNAcI, representing cultures 1through 5:

TABLE 8 Pellet and Inclusion Body Weights from 150 ml JM109 pCWin2MBP-ST6GalNAcl Cultures JM109 pCWin2 MBP- Inclusion ST6GalNAcI CellPellet Weight Body Weight Constructs (g) (g) D32 0.65 0.30 E52 0.98 0.73S127 0.56 0.57 S186 1.2 0.93 S201 1.1 0.83

Pellet weights and inclusion body weight were determined for cultures in1 L shaker flasks and 1.5 L fermenters including JM109 pCWinMBP-ST6GalNAcI constructs S186 and S201 cultures. Protein samples werediluted and concentration was measured at OD₂₈₀. Protein concentrationand percent of solubilized protein conversions were calculated for allfive truncated ST6GalNAcI clones, as set forth in Table 9.

TABLE 9 Pellet and Inclusion Body Weights from 1 L Shaker flasks and 1½L Fermenters JM109 pCWin2 MBP-ST6GalNAcI Cultures JM109 pCWin2 MBP-Inclusion ST6GalNAcI Cell Pellet Weight Body Weight Constructs (g) (g)S186 Shaker flask 10.2 2.30 S201 Shaker flask 8.22 2.94 S186 Fermenter14.33 1.47 S201 Fermenter 12.48 2.67 Protein Concentration and %conversion of 150 ml. JM109 pCWin2 MBP-ST6GalNAcI cultures afterSolubilization. JM109 pCWin2 Protein MBP- Con- Protein ProteinST6GalNAcI A₂₈₀ After centration % of Concentration ConstructsSolubilization (mg/ml) Conversion (mg/ml) D32 0.113 4.56 3.9 1.0 and 0.1E52 0.129 5.00 2.5 1.0 and 0.1 S127 0.153 5.03 5.5 1.0 and 0.1 S1860.201 5.68 2.3 1.0 and 0.1 S201 0.274 9.93 12.4 1.0 and 0.1

TABLE 10 Protein refolding conditions used with the Hampton ResearchFoldit kit

TABLE 11 Results from initial refold buffer screen. In this assay, allfive constructs were tested under all 16 refold conditions from theHampton Foldit kit (Hampton Research, Aliso Viejo, CA). These refoldswere purified by G- 50 gel filtration and then tested for activity bythe radioactive assay as described above. Refold condition 1 2 3 4 5 6 78 Raw CPM D32 78 38 131 54 53 44 47 160 E52 142 165 346 155 136 178 133152 S127 126 345 381 267 238 186 247 166 S186 341 373 779 335 289 180337 386 S201 3387 2892 3496 1566 2077 1580 2851 5186 Sf9 + Control 1942Negative 93 Control Corrected CPM D32 42 2 95 18 15 8 11 124 E52 38 61242 51 32 74 29 48 S122 −64 155 191 77 48 −4 57 −24 S186 300 332 738 294248 139 296 345 S201 1382 887 1491 −439 72 −425 846 3181 % CPM D32 0.080.00 0.19 0.04 0.03 0.02 0.02 0.25 E52 0.05 0.08 0.32 0.07 0.04 1.000.04 0.06 S127 −0.08 0.18 0.23 0.09 0.06 0.00 0.07 −0.03 S186 0.37 0.410.90 0.36 0.30 0.17 0.36 0.42 S201 1.64 1.06 1.78 −0.52 0.09 0.051 1.013.79 Refold condition 9 10 11 12 13 14 15 16 Raw CPM D32 67 46 160 35150 79 39 32 E52 286 158 298 226 178 150 367 205 S127 196 125 268 274210 149 159 216 S186 330 302 1795 447 289 465 2476 358 S201 7665 10993158 2932 2585 871 2559 2343 Sf9 + Control Negative Control CorrectedCPM D32 31 10 124 −1 114 −7 3 −4 E52 182 54 194 122 74 46 263 101 S122 6−65 78 84 20 −41 −31 26 S186 289 261 1754 406 248 424 2435 317 S201 5660−906 1153 927 580 1134 554 338 % CPM D32 0.06 0.02 0.25 0.00 0.23 −0.010.00 0.00 E52 0.24 0.07 0.26 0.16 1.00 0.06 0.35 0.13 S127 0.00 −0.080.09 1.00 0.02 −0.05 −0.04 0.03 S186 0.35 0.32 2.15 0.50 0.3 0.52 2.90.39 S201 6.74 −1.08 1.37 1.1 0.69 −1.35 0.66 0.40

Results from this radioactive assay indicated that refold conditions 8and 9 worked best for construct S201. Two conditions—8 and 11—forconstruct S186, condition 12 for construct S127 and condition 6 forconstruct E52 provided the highest CPM count.

TABLE 12 Results from S201 Construct Refold # 8 and # 9. In this assay,construct S201 was re-tested under refold conditions 8 and 9 with 1.0and 0.1 mg/ml concentration with and without DTT from the Hampton Folditkit (Hampton Research, Aliso Viejo, CA). The refolded proteins werepurified by G-50 gel filtration and then tested for activity by theradioactive assay. Results indicate that Refold # 8 holds higher CPMcounts than refold # 9. ¹⁴C Activity Foldit Screen ST6GalNAcI/S201 AssayRaw Beffer # pH Tris MES Detergent Polar/Non DTT GSH/GSSG mg/ml CPMCorr.CPM % CPM 8-1 6.5 − + + Arginine − +/+ 1.0 4570 2678 3.19 8-2 6.5− + + Arginine − +/+ 0.1 3296 1404 1.67 9-1 6.5 − + + Sucrose − +/+ 0.12192 561 0.67 9-2 6.5 − + + Sucrose − +/+ 1.0 1472 −159 −0.19 NA + 8-11892 NA + 9-2 1631 A-Enz 2560 NA/NE 4397 Cont. = 2 μl 84000 Blank NA =No NE = No A-E = Acceptor- AM644- 27 Acceptor Enzyme Enzyme pg46

TABLE 13 Results of the repeat of Refold # 11 of S186 and Refold # 8 ofS201. These proteins were used to analyze transfer of Sialic acid toG-csf-O- GalNac(AM644-pg150-156). ¹⁴C Activity Foldit ScreenST6GalNAcI/S186 Assay Raw Corr. Beffer # pH Tris MES Detergent Polar/NonDTT GSH/GSSG mg/ml CPM CPM % CPM 11-1 8.2 + − − Arginine − +/+ 1.0 520484 0.6 11-2 8.2 + − − Arginine − +/+ 0.1 695 581 0.7  8-1 6.5 − + +Arginine − +/+ 0.1 784 748 0.8  8-2 6.5 − + + Arginine − +/+ 1.0 206 1700.2 BV + Cont 2392 2356 2.7 −Acp/ 36 +Enz +Acp/−Enz 54 Cont. = 2 μl88491 Blank NA = No NE = No A-E = Acceptor- AM644-pg46 29 AcceptorEnzyme Enzyme

From results obtained in the screening process, it was determined thatrefold conditions 8 and 9 for S201 and conditions 8 and 11 for S186yielded the most promising results. To achieve reproducibility,additional refolding reactions were performed under the same conditionsusing G-50 gel filtration for refolds 8 and 9 for S201 and 8 and 11 forS186. From these experiments, refold 8 yielded higher counts and wasfound to be reproducible while conditions 9 and 11 did not.

A granulocyte-colony stimulating factor (G-CSF) assay was performed withrefolded proteins of constructs S186 and S201 in refold buffer 8 TheG-CSF reaction was allowed to incubate at 32° C. for 5 days. Thereaction was analyzed at 1, 2 and 16 hours and at 1, 2 and 5 days timepoints. The parental peak for GalNAc−O-G-CSF is expected at MW ˜19006. Asuccessful reaction is indicated by addition of ˜309 and 509 molecularweight to that peak. From the 5 days data for refolds S201 a developingpeak was seen at ˜19313 (GalNAc+SA) and 19515 (2GalNAc+SA), a differenceof approximately 307 and 509. This data again illustrated that sialicacid was added to GalNAc−O-G-CSF by the refolded truncated mouseST6GalNAcI proteins and confirmed what was reported by the radioactiveassay.

These data support the conclusion that refolded E. coli-expressedMBP-ST6GalNAc 1 is an active sialyltransferase enzyme, and that underrefold condition 8 found in Hampton Research's Foldit kit (HamptonResearch, Aliso Viejo, Calif.), active conformations of MBP-ST6GalNAcIconstruct S201 (Δ200) are achievable. The generation of a functionalrefolded protein was demonstrated in the [14C] radioactive andGalNAC−O-G-CSF assays.

Example 3 Cloning and Expression of Human and Mouse GalNAcα-2,6-Sialyltransferases (ST6GalNAcI) in a Baculovirus Expression System

The expression of both human and mouse GalNAc α2,6-sialyltransferases(ST6GalNAcI) was demonstrated in Sf9 (insect) cells. To examine theexpression of human GalNAc α-2,6-sialyltransferase (hST6GalNAcI) in Sf9(insect) cells, the long form of full-length human cDNA was constructedby PCR cloning of two EST clones into pcDNA3.1(+)(GenBank accessionnumber is Y11339; there is a shorter form of cDNA in the NCBI database). Three truncated forms of hST6GalNAcI, K36, K125 and S258(corresponding to Δ35, Δ124 and Δ257) were cloned into the baculovirusvector pAcgp67B based on this hST6GalNAcI clone. All three truncationscan be expressed in Sf9 cells and K36 showed the highest activity. Amouse ST6GalNAcI in a baculovirus expression vector in pAcgp67A calledpTS103 (K31 truncation, corresponding to Δ30) was also obtained. Twoadditional truncations, S127 and S186 (corresponding to Δ126 and Δ185)were made and expressed in the baculovirus vector pFastBac-1-gp(Invitrogen, Carlsbad, Calif.). Expression studies on these threetruncations showed that S127 has the highest expression level.

Described herein are the processes of cloning and expression of bothhuman and mouse GalNAc α-2,6-Sialyltransferases (ST6GalNAcI) in Sf9(insect) cells, including the source of cDNAs, detail description ofsteps in the assembly of final expression plasmid, the expression andthe enzymatic activities of the secreted proteins.

Two human EST clones containing two fragments of human ST6GalNAcI (theclone IDs are 4816713/Cat#97002RG and 6300955/Cat#97002RG) were obtainedfrom invitrogen. These clones were obtained as bacterial glycerol stocksin tubes on dry ice. The bacterial stocks were streaked on a LB agarplate containing ampicillin for clone #4816713 and on a LB agar platecontaining chloramphenicol for clone# 6300955. The plates were incubatedat 37° C. overnight. Three individual colonies were picked andinoculated into 5 ml LB culture. DNA plasmid was isolated using QIAprepSpin Miniprep Kit (Qiagen, Valencia, Calif.). Enzymatic digestionsshowed that clone #4816713 has an insert of about 2-2 Kb released byEcoRI and clone# 6300955 has an insert of about 1.5 Kb released by EcoRI and Xho I (FIG. 1). Both clones released the expected sizes ofinserts.

By comparing the sequences to the published human ST6GalNAcI (long form,accession# Y11339), it is clear that clone #4816713 covers the entiresequence except a fragment from nucleotide #f 1375 to 1480. Clone#6300955 covers sequences from nucleotide #1070 to the C-terminus.Therefore, two sets of PCR primers were designed for cloning the fulllength human ST6GalNAcI cDNA. The first set of primers is: hST6GN1-F1,caGGATCCacatgcagaaccttcc (SEQ ID NO:49) and hST60N1-R2,gtcccgggtcgccttccaggaagtgeaagtagcggacgtccttcccaagaggcacg (SEQ ID NO:50).The second set of primers is: hST6GN1-F2, ggaaggcacccgggac (SEQ IDNO:51) and hST6GN1-F1, ccGAATTCcggtcagttcttggct (SEQ ID NO: 52) (capitalletters represent the restriction sites BamH I and EcoR I for cloninginto pcDNA3.1, and the underlined residues indicate the XmaI site in thecDNA for putting the two pieces together).

The N-terminal fragment of hST6GalNAcI was amplified using clone#4816713 DNA as template, the first set of primers discussed above andPfu DNA polymerase. The C-terminal fragment of hST6GalNAcI was amplifiedusing clone# 6300955 as template, the second set of primers and pfu DNApolymerase. The PCR fragments were gel-purified using QIAEX II gelpurification kit (Qiagen, Valencia, Calif.). Both DNA fragments werecloned into pCR-Blunt vector (Invitrogen, Zero Blunt PCR Cloning Kit,Carlsbad, Calif.). EcoR I digestions showed that both pCR-hST6-N#1-6 andpCR-hST6-C#1-6 have correct insert size.

pCR-hST6GalNAcI-N#1 and pCR-hST6GalNAcI-C#1 were digested with BamH Iand Xma I, and Xma I and EcoR I, respectively. The released fragmentswere ligated with pcDNA3.1 (+) cut with EcoRI and BamHI. The finalproduct pcDNA3.1 (+)-hST6GalNAcI-N1C1#1 was confirmed by both enzymaticdigestions and DNA sequencing analysis. The obtained hST6GalNAcI cDNAhas three nucleotide changes and two of them change the amino acidsequences (Q65K and M3791) These differences all originated from the ESTclones

Three additional primers were designed to generate 3 truncations ofhST6GalNAcI for expression in Sf9 cells. The primers are: bST6-K36-5′,ccaGGATCCaaggagcctcaaac (SEQ ID NO:53), hST6-K125-5′,ccaGGATCCaagagcccagaaaaagag (SEQ ID NO:54), and hST6-S258-5′,ccaGGATCCtctgagcctcggtgg (SEQ ID NO:55) (capital letters represent therestriction site BamH I for cloning into pAcgp67B). The K36 clone istruncated immediately after the transmembrane domain of human ST6GalNAcIand the S258 clone is truncated at the same relative position as thechicken ST6GalNAcI T233, according to an amino acid sequence comparison.The latter is the same published truncation used for chicken ST6GalNAcIexpression in Sf9 (Kurosawa, N., et al (1994) J. Biol. Chem. 269,1402-1409).

Three PCR products were obtained using the three primers paired withhST6GN1-R1, pcDNA3.1 (+)-hST6GalNAcI-N1C1# 1 as template and pfu DNApolymerase. All were cloned into pCR-blunt. K36#6, K125#4 and S258#6sequence analysis confirmed that the vectors contained the correctcDNAs. The inserts from the pCR-blunt vector were cloned into the BamHIand EcoRI sites of pAcgp67B in-frame with the gp67 signal sequences. Thesequences of the three trunctations, pAcgp67B-K36#4, K125#4 and S258#2were confirmed by DNA analysis and were identified as the same as thefull length human ST6GalNAcI sequences.

The DNA of above three truncated hST6GalNAcI in pAcgp67B, K36, K125 andS258, were co-transfected with BaculoGold DNA using BD BaculoGoldTransfection Kit (BD Bioscience, Franklin Lakes, N.J.). To amplify thebaculovirus, 0.1 ml of the transfection supernatant was used to infect10 ml of Sf9 cells at 2×10⁶ cells/ml in a 10-cm dish. The P1 supernatantwas harvested 3 days after infection. P2 viral stock was obtained byinfecting 50-ml Sf9 cells at 2×10⁶ cells/ml and MOI=0.2. The baculovirussupernatants were amplified twice to get high titers. The virus titerswere determined by BacPAK Baculovirus Rapid Titer Kit (BD Bioscience,Franklin Lakes, N.J.). A 50-ml scale production was set up at MOI=2,2×10⁶ cells/ml. The culture supernatants were obtained at day 2-4. AST6GalNAcI assay showed that both K36 and K125 expressed at 0.25-0.35U/liter and S258 at 0.1-0.2 U/liter at 50-ml scale. Twelveplaque-purified K36 clones were further tested and amplified. Clone# 10demonstrated the highest activity (1 U/liter). 1-liter scale productionof clone# 10 had an expression level at 3 U/liter

pTS103 DNA (10 μg) was transformed into TOP10 cells and DNA wassubsequently prepared from single colonies using a QIAprep Spin MiniprepKit (Qiagen, Valencia, Calif.). pTS103 was analyzed by DNA sequencinganalysis and the data demonstrated that this clone has severalnucleotide differences from the published sequences. pTS103 is pAcgp67Awith mouse ST6GalNAcI (mST6GalNAcI) having a K3 truncation and a myc tagat the end of C-terminus in between BamH I and BgI It restriction sites.

Primers were designed for making truncated mST6GalNAcI: S127 and S186.The primers were: S127-EcoRI-5′, cgGAATTCtctcagaacacctggac (SEQ IDNO:56), S186-EcoRI-5′, cgGAATTCtctctgagcctcggtgg (SEQ ID NO:57,mST6-XhoI-3′, gcCTCGAGtcagttctttgctttgtc (SEQ ID NO 58) (Capital lettersrepresent the restriction sites for EcoR I and XhoI). The cloning vectorused was pFastBac-1-gp, from Invitrogen (Carlsbad, Calif.), and a gp67signal sequence was inserted between BamH I and EcoR I sites.

Two PCR products were obtained using the three above-referenced primers.pfu DNA polymerase and pTS103 were used as a template and cloned intopCR-blunt. Sequence analysis confirmed that pCR-S127#2 and S186#2contained the correct cDNAs. The inserts from the pCR-blunt vector werecloned into the EcoR I and Xho I sites of pFastBac-1-gp in-frame withthe gp67 signal sequences. pFastBac-1-gp-S127#3 and S186#2 wereconfirmed by EcoRI and XhoI double digestions and DNA sequence analysis.

pFastBac-1-gp-S127#3 and S186#2 DNA were transformed into DH10Baccompetent cells from the Bac-to-Bac Baculovirus Expression System(Invitrogen, Carlsbad, Calif.). 12 white colonies from eachtransformation were re-streaked on plates and 8 out of 12 were actuallywhite in color. “Bacmid” DNA was isolated using P1, P2 and N3 bufferswith QIAprep Spin Miniprep Kit, according to the protocol from themanual (Qiagen, Valencia, Calif.). PCR screening was conducted to detectthe insert of mST6GalNAcI in the bacmid DNA using M13F and mST6-XhoI-3′as primers and Taq DNA polymerase (Qiagen, Valencia, Calif.). All 8clones from each construct have the correct inserts and they were thesame as the pTS103 sequences.

Additional bacmid DNA of S127, clone #5 and 6, S186, clone#3 and 4 wereisolated from the bacteria using S.N.A.P MidiPrep Kit (Invitrogen,Carlsbad, Calif.). The bacmid DNA was tranfected into Sf9 cells usingCellfectin (Invitrogen, Carlsbad, Calif.). The baculovirus supernatantswere amplified once to obtain high titers. The virus titers weredetermined by BacPAK Baculovirus Rapid Titer Kit (BD Bioscience,Franklin Lakes, N.J.). A 50-ml scale production was set up at MOI=2,2×10⁶ cells/mt. The culture supernatants were obtained at days 2-4.ST6GalNAcI assay showed that both S127 viral stocks produced higheractivities at 0.15-0.25 u/liter at 50-ml scale than either S186 viralstocks. Twelve plaque-purified S127 clones were further tested andamplified. All clones demonstrated the same activity, but clone#4 hadslightly higher activity (0.46 u/liter). One-liter scale production ofclone#4 demonstrated an expression level of 1.7 u/liter.

The above work demonstrated that both human and mouse GalNAcα-2,6-sialyltransferases (ST6GalNAcI) can be expressed in Sf9 (insect)cells and that the enzymes were secreted into the culture medium, withan expression level of about 2-3 u/liter.

Example 4 Expression of chickenN-acetylgalactosamine-α2,6-sialyltransferase (ST6GalNAcI) in Sf9 CellsUsing Recombinant Baculovirus

Chicken N-acetylgalactosamine-α2,6-sialyltransferase (ST6GalNAcI) wasexpressed in Spodoptera frugiperda (Sf9) cells using the baculovirusexpression vector system. N-acetylgalactosamine-α2,6-sialyltransferase(ST6GalNAcI) transfers sialic acid from CMP-sialic acid by an α2,6linkage onto the C-6 hydroxyl group of a N-acetylgalactosamine (GalNAc)residue.

This enzyme was produced by infecting cultures of Sf9 cells withrecombinant baculovirus. An alternate non plaque-purified baculovirusstock of chicken ST6GalNAcI was also used, based on use of the alternateclone in the published literature. This alternate clone was previouslythought to be truncated at amino acid T233, but N-terminal sequenceanalysis showed that an extra amino acid before T233 was introducedduring cloning, and, therefore, the polypeptide produced by thealternate clone contains amino acid K (lysine) 232 from the full lengthST6GalNAcI sequence. Therefore, the alternate clone is actuallytruncated at K232. This stock was plaque-purified, amplified, andsubsequently used for experiments herein.

Described herein are experiments conducted to obtain baculoviral DNAfrom plaque-purified viral stocks of the chicken ST6GalNAcI for sequenceanalysis of the enzyme and the conditions used to produce the enzymefrom these viral DNA stocks. In this study, the enzyme produced had anaverage expression level of 11.8 units/L when produced in I liter scaleusing the following conditions: MOI=5-10, 130 rpm, 27° C., total cellcount of 3.5e⁹ cells-7e⁹ cells and 72 hours of incubation.

Baculovirus DNA was isolated according to the following protocol. To theconcentrated virus stock was added 6 μl 0.5 M EDTA and 4.5 μl 1 MTris-HCl, pH 8.0. Then, 0.3 ml lysis buffer (0.2 M NaOH, 1% SDS) wasadded and the mixture incubated at room temperature for 5 minutes. Afterlysis, 0.3 ml of neutralization buffer (3M NaOAc, pH 5.2) was added andthe mixture was incubated at 4° C. for 10 minutes. The mixture wasclarified by centrifugation at 14,000 rpm for 10 minutes, at 4° C., in amicrocentrifuge. The baculovirus DNA in the resulting 0.84 mlsupernatant was precipitated using 0.8 ml isopropanol and incubated onice for 10 minutes. The precipitated virus DNA was collected bycentrifugation at 14,000 rpm for 10 minutes at room temperature. Theresultant DNA pellet was washed with 0.5 ml 70% ethanol and air dried.The DNA pellet was then dissolved in 20 μl 1×TE Buffer (10 mM Tris-HCl,1 mM EDTA, pH 8.0). DNA concentration was measured using OD260. TheOD₂₆₀ 0.012, and therefore, DNA concentration=600 μg/ml.

Isolation of the chicken ST6GalNAcI was conducted using PCR. The primersused included ch233BamHI2 5′-GAT TCG GGA TCC ACG GAG CCA CAG TGG GAT TTTG-3′ (SEQ ID NO:59) and ch233Xho13′-GAT CGC CTC GAG TCA GGA TCT CTG GTAGAG CTT C-3′(SEP ID NO:7). A PCR reaction was set up with the followingcomponents: 5 μl 10×PCR Buffer, 2 μl 10 mM dNTP, 1 μl 5′ primer (10pmol/μl), 1 μl 3′ primer (10 pmol/μl), 2 μl DMSO, 1 μl DNA template, 0.5μl Herculase enzyme (Stratagene, Carlsbad, Calif.), and 37.5 μl PCRgrade H₂O. The PCR program conditions included cycles of 95° C., 3minutes; 95° C., 45 sec; 42° C., 1 minute, 72° C. 1 minute for 5 cycles;95° C., 45 sec; 57° C., 1 minute, 72° C. 1 minute for 35 cycles; 72° C.,10 minutes; 4° C. pause.

PCR products were isolated using a MinElute Gel Extraction Kit (Qiagen,Valencia, Calif.). The DNA was eluted in 20 μl 1×TE (10 mM Tris-HCl, 1mM EDTA, pH 8.0). pCRBlunt ligation and transformation was conductedusing 4 ti of the PCR reaction product, 1 μl salt solution, and 1 μlTOPO pCR4 Blunt vector (ZeroBlunt TOPO, Invitrogen, Carlsbad, Calif.). Avolume of 6 μl of the ligation mixture was then added to 50 μl of Top10cells. The following ligation incubations were performed: First, on icefor 30 minutes, at 37° C. for 1 minute, then, on ice for 2 minutes.Reactions were conducted by adding 0.5 ml SOC medium, then incubatingthe mixture at 37° C. for 1 hour. After incubation, 200 μl of themixture was plated on a Kanamycin-containing plate. About 100 colonieswere generated.

Direct cloning of the PCR products was also carried out under thefollowing conditions. A reaction mixture included 16 μl PCR product, 1μl BamHI, 1 μl XhoI, 4 μl BamHI Buffer, 20 μl H₂O. The reaction mixturewas incubated at 37° C. for 2 hours. Another reaction mixture included 1μl pCWIN2-MBP vector (0.35 mg/ml), 0.5 μl BamHI, 0.5 μl XhoI, 2 μl BamHIbuffer, and 16 μl H₂O. The reaction mixture was incubated at 37° C. for2 hours. After gel electrophoresis of the direct cloning products, gelextraction, ligation and transformation, 10 colonies were selected andgrown for plasmid DNA minipreps. Out of these 10 minipreps, 6 containedthe correct insert in a pCWIN2-MBP vector.

Chicken ST6GalNAcI truncated at amino acid 232 was expressed andproduced in Sf9 cells at a 1 liter scale using recombinant baculovirus,using conditions including a 1 liter scale, MOI=5-10, 130 rpm, 27° C.,total cell count of 3.5e⁹ cells-7e⁹ cells and 72 hours of incubationtime. The average expression level of the enzyme in these productionruns is 11.8 Units/L.

Cells were counted using the Hemacytometer Method, and a workingsolution of trypan blue was prepared. Trypan blue is initially a 0.4%solution and it is diluted with PBS to a working concentration of 0.04%(1:10 dilution). A sample of cell suspension was aseptically withdrawnto be counted and dilutions (1:2, 1:4, 1:5, 1:10, 1:20) were prepared,as necessary, in the trypan blue working solution. Cells were countedwithin 3 minutes after being stained with trypan blue. Approximately 10μl of the stained cell suspension was withdrawn and the tip of the pipetwas placed onto the slot of a clean hemacytometer with coverslip. Thecell suspension passed under the coverslip by capillary action. Thehemacytometer was placed on the stage of an inverted microscope andread. The viable cell count was determined by using the equation: ViableCell (Cells/ml)=(Number of Viable Cells Counted)/(Number of SquaresCounted)×104× Dilution Factor. That is, the total viable cell number inthe original suspension was found by multiplying the viable cells/ml bythe total ml in the original suspension.

A plaque purification assay was then used. The method included countingSf9 cells and determining viability, as described above. Cells must beat least 90% viable and in log phase growth. Cells were diluted withfresh media to a density of 5e⁵ cells/ml with a final volume between 20and 30 ml. A volume of 2.0 ml of the cell suspension was added to eachwell in two 6 well plates and cells were rocked to distribute cellsevenly. Each well contained approximately 1e⁶ cells. Plates were placedin a sealed container containing 2 paper towels dampened withapproximately 50-100 ml of water to provide humidity. Plates were thenplaced on a rack on top of the towels to prevent direct contact with thewet towels, and were incubated in the container at 27° C. for 1-4 hoursuntil the cells adhered to the bottom of the wells. Serial dilutions of1:10 of the virus stock were made, from 1.0 e- to 1.0 e⁻⁹. A volume of0-5 ml virus stock was placed into 4.5 ml SFM Sf-900 II media fordilution of the stock.

When the cells formed an even monolayer of about 70-80% confluency, themedia was aspirated from the cells using a sterile pipette. A negativecontrol was prepared by gently adding 1 ml of fresh media to each of twowells. Two wells for each dilution were infected, from 1.0 e⁻² to 1.0e⁻⁹, by gently adding 1 ml of the virus dilution to each well. Theplates were incubated at room temperature for 1 hour on a level surfaceto allow the virus to infect the cells. Plaquing medium was thenprepared in a sterile 100 ml bottle, containing 30 ml of Sf-900 II 1.3×in 10 ml of 4% agarose. The bottle was incubated in a 37° C. waterbathuntil ready to use (after 1 hour viral incubation). After the 1 hourincubation, the virus inoculum was aspirated from the cells using asterile pipette by tilting the plate and aspirating from the edge. 2.0ml of plaquing medium was added to each well. The agarose was allowed toset for 10-15 minutes at room temperature, then the preparations wereincubated at 27° C. in the sealed container with wet paper towels for 5to 7 days, until the plaque appeared.

Plaque purification was conducted by picking a plate with plaques thatwere spaced far apart. Using a sterile Pasteur pipet and bulb, a clearplaque was picked and transferred, via agarose plug (containing virus),to a sterile 1.5 ml microcentrifuge tube containing 500 μl SFM Sf-900 IImedia. The agarose plugs were incubated in media at 4° C. overnight.Virus was amplified to Passage 1 (P=1) amplification.

Six-well plates were seeded with log-phase Sf9 cells at 7e⁵ cells/ml in3 mls (˜2.0e⁶ cells total/well) and allowed to settle for 5-15 minutesat room temperature. Plates were infected with 100 μplaque “pick-up” andshaken gently. One well with no infection was used as a negativecontrol. Plates were incubate at 27° C. for 3-4 days, until observationof signs of infection (grainy-looking, shriveling, dying cells).Supernatant was harvested and assayed for protein. The well containingthe highest activity for further amplification was the P=1 virus stock.

Asialo Bovine Submaxillary Mucin (asialo BSM) or asialo OvineSubmaxillary Mucin (asialo OSM) substrate was prepared for a ST6GalNAcIenzyme assay. Sialic acid was released by hydrolysis, in a reactioncontaining 500 μl BSM or OSM (20 mg/ml), 500 μl dH₂O, and 130 μl 2 Mglacial acetic acid. Components were mixed and incubated at 80° C. for 5hours to 18 hours. The reaction mixture was diluted with 5 ml PBS.Samples were loaded onto Amicon Ultra-15 columns and centrifuged at3,000×g 4° C. for 20 minutes (Millipore, Bedford, Mass.). Five ml of PBSwas added and the columns centrifuged again. The process was repeatedthree times, or until the mixture was at approximately pH 7.0. UntreatedBSM or OSM was used to prepare a standard curve to estimate theconcentration of AOSM or ABSM by linear regression.

A radioactive assay was used to assay ST6GalNAcI. The reaction mixtureincluded CMP 14C sialic acid (dried down by nitrogen) at a concentrationof 100,000 CPM, cold CMP sialic acid at 0.2 mM (10 nmoles total inreaction), A-BSM (acceptor substrate, 0.25 mg), MES pH 6.0 at 50 mM, andNaCl at 100 mM, with 10 μl of enzyme sample in a total of 40 μl reactionvolume. Enzyme-free and/or acceptor-free negative control(s) wereincluded. The reaction mixtures were incubated at 37° C. for 1 hour, atwhich point, 100 μl (per reaction) of 5% phosphotungstic acid/15% TCAwas added and mixed well. The sample was prepared by centrifugation atmaximal speed in microfuge for 2 minutes and the supernatant discarded.TCA (5%) was added at 500 μl per reaction and the sample vortexed. Thesample was again centrifuged at maximal speed in a microfuge for 2minutes, the supernatant discarded by pipetting. Pellets wereresuspended in 100 μl 10N NaOH, 1 ml water was added, and 5 mlscintillation fluid was added to the resulting mixture, and the mixturecounted for 1 minute.

TABLE 14 Calculations for ST6GalNAcI activity in Units/Liter. Unit =transfer of 1 μmol of CMP Sialic Acid/minute U/L = [(cpm corr) (DF) (10nmoles CMP sialic acid) (1 umol) (1000 μl) (1000 ml) (5.5 conversionfactor)]/[(total cpm corr) (60 min) (10 μl sample volume) (1000 nmol) (1ml) (L)] background cpm = cpm of sample with no enzyme or no acceptorcpm corr = cpm minus background cpm total cpm corr = total cpm minusbackground cpm Conversion factor = Factor for working at a acceptorsubstrate concentration less than the Km as determined by previousrelated work.

Passage 2 viral amplification was conducted by growing suspension of Sf9cells to a concentration of 2.0 e⁶ cells/ml in 250 ml disposableerlenmeyer flask, which contained 30 ml to 50 ml of SFM Sf-900 II mediaTitered viral stock was added at an MOI of 0.2, and fresh SFM Sf901media was added to a total volume of 50 ml to 100 ml. The cultures wereincubated in shaking incubator for 48 hours, at 27° C., 130 rpm. Cellswere harvested by centrifugation using sterile 250 ml conical centrifugetubes. The viral stock was titred by end point dilution assay.

Large scale virus stock was prepared in 59 cells. A suspension of S59cells was grown to a concentration of 7.0 e⁶ cells/ml to 1.4 e⁷ cells/ml(3.5e⁹ to 7e⁹ total cells) in a 2 L non-baffled fernbach flaskcontaining 500 ml of SFM Sf-900 II media. Titered viral stock was addedat an MOI of 0.2, and fresh SFM Sf900II media was added to a totalvolume of 1 liter. The cultures were incubated in a shaking incubatorfor 48 hours, 27° C., 130 rpm, and the cells harvested by centrifugationusing sterile 1 L centrifuge bottles. The viral stock was titred by anend point dilution assay and stored at 4° C.

Viral stocks were also titred using and end point dilution assay asfollows. Cells were counted and viability determined as described above.Cells were at least 90% viable and in log phase growth. Cells werediluted with fresh media to a density of 2.5e⁵ cells/ml in 10 ml andcells were then plated at 10 μl/well in 72-well microtiter plate. Mediawas plated only in the last 2 wells of each row. Serial (1:10) dilutionsof virus stock from 1.0 e⁻¹ to 1.0 e⁻⁹. Virus stock (100 μl) was placedinto 900 μl SFM Sf-900 II media for dilution (1.0 ml volume totaldilution), and 10 μl of the 1.0 e⁻¹ diluted stock was placed into eachof 10 wells of the first plate. Plates were incubated at 27° C. for 7days in a humid container. The plates were observed using a microscopewith a 10× objective. Wells were scored as “infected” or “not infected.”The Reed-Muench formula (Reed, L. J., and Muench, H. (1938), Amer. Jour.Hygiene, 27, 493-497.) was used to determine 50% infectivity dose(TCID₅₀) of virus is used to determine viral titer. FIG. 25 illustratesthe titer determination worksheet used as described above.

Large scale protein ST6GalNAcI production in Sf9 Cells included growinga suspension of Sf9 cells to a concentration of between 7.0 e⁶ cells/mlto 1.4 e⁷ cells/ml (3.5e⁹ to 7e⁹ total cells) in 2 L non-baffledfernbach flask containing 500 ml of SFM Sf900II media. Titered viralstock was added to the culture at an MOI of 5-10. Fresh SFM Sf900IImedia was then added to a total volume of 1 liter, and the culturesincubated in shaking incubator for 72 hours, at 27° C., 130 rpm. Cellswere harvested by centrifugation using sterile 1 Liter centrifugebottles. The resultant supernatant was filtered through a 0.21 um filterunit and the final product stored at 4° C.

TABLE 15 ST6GalNAcI activity of screening plaque-purified P = 1 viralstocks Corrected ST6GalNAcI Sample Sample cpm Sample cpm DF activity U/LBlank (NaOH only) 10 — 1 — Blank (no enzyme) 23 — 1 — Blank (media only)23 — 1 — Blank (no acceptor) 21 — 1 — ch-ST6GalNAcI pure 9744 9724.75 10231.523 ch-P1 Clone #1 42 22.75 1 0.054 ch-P1 Clone #2 121 101.75 10.242 ch-P1 Clone #3 62 42.75 1 0.102 ch-P1 Clone #4 168 148.75 1 0.354ch-P1 Clone #5 121 101.75 1 0.242 ch-P1 Clone #6 67 47.75 1 0.114 ch-P1Clone #7 153 133.75 1 0.318 ch-P1 Clone #8 116 96.75 1 0.230 ch-P1 Clone#9 71 51.75 1 0.123 ch-P1 Clone #10 158 138.75 1 0.330 ch-P1 Clone #1255 35.75 1 0.085 ch-P1 Clone #13 69 49.75 1 0.118 ch-P1 Clone #14 7555.75 1 0.133 ch-P1 Clone #15 61 41.75 1 0.099 ch-P1 Clone #16 49 29.751 0.071 Average blank cpm 19.25 Total cpm 50834

The purpose of screening the plaque-purified P=1 viral stocks is toidentify a single clonal isolate containing enzyme activity. Clone 4(0.354 U/L), Clone 6 (0.318 U/L), and Clone 10 (0.330 U/L) had thehighest activities and are good candidates for further amplification.Clone 4 was chosen since it had the highest activity of the three.

TABLE 16 Large-scale production of chicken ST6GalNAcI Total cellActivity density Production Harvest (Units/ Production Lot # MOI atinfection Scale (L) Time (hrs) Liter) 4-081503-1LP1 10 4.5e9 cells 1 729 4-81903-1LP2 10 3.6e9 cells 1 96 0 4-82603-1LP3 10 5.1e9 cells 1 72 94-82803-1LP4 5 5.5e9 cells 1 72 8 4-90203-1LP5 8.3 4.6e9 cells 1 72 124-90903-1LP6 10   7e9 cells 1 96 2 4-91603-1LP7 10 5.5e9 cells 1 72 10492203-1LP8 10 3.5e9 cells 1 72 23

The sequence of Chicken ST6GalNAcI was confirmed as follows. N-terminalsequencing was conducted using 20 ug of purified chicken ST6GalNAcI,resulting in the sequence: VSTEDPKTEPOWDFDDEYILDSSS (SEQ ID NO:8), whichverified that the chicken ST6GalNAcI used for the experiments describedherein had the same amino acid sequence (underlined) as published X74946chicken ST6GalNAcI truncated at amino acid K232. DNA sequencing of thechicken ST6GalNAcI was conducted using 50 ml of chicken ST6GalNAcIbaculovirus stock. Viral DNA was extracted from this stock,PCR-amplified, inserted into the vector pCWIN2-MBP, and sequenced. DNAwas sequenced from the point of the T233 truncation, not the K232truncation. The resulting DNA had Sac2/Kpn2 restriction sites, and had1029 bases with a 49.36% GC content (FIG. 26). Translation of thesequence obtained, shown in FIG. 27, revealed a one residue differencewhen compared to published chicken ST6GalNAcI GenBank X74946, namely,V25 IA (GTA to GCA, valine to alanine). The experimental DNA sequencehad one other mutation, a silent mutation T233 (ACT to ACG, same aminoacid, threonine) in pCWIN2-MBP-chST6GalNAc, which was introduced by aPCR primer during cloning.

K232 was not included in when viral DNA was PCR amplified. The rest ofthe DNA sequence was verified to be the same as the published sequence.

In summary, chicken ST6GalNAcI viral stock was plaque-purified,amplified, and enzyme was produced from the stocks. Eight productionruns were done at a 1 liter scale. Two of the runs that were infectedfor 96 hours had little or no activity. The best conditions seen for theproduction runs performed during the time of this report are MOI=5-10,130 rpm, 27° C., total cell count of 3.5e⁹ cells to 7e⁹ cells, and 72hours of incubation. Under these conditions, the average activity of theproduced ST6GalNAcI was 11.8 units/liter. The chicken ST6GalNAcIsequence was also verified N-terminal sequencing was performed onpurified chicken ST6GalNAcI protein and sequence analysis confirmed thatit was truncated at E32 and had the same amino acids in the N-terminalportion as the published sequence DNA sequencing was also performed forverification of sequence. Recombinant viral DNA was extracted fromchicken ST6GalNAcI baculovirus stock and PCR amplified. The DNA was PCRamplified from the T233 truncation and not K232. The DNA was insertedinto vector pCWIN2-MBP and sequenced. Results revealed one basedifference (GTA to GCA) in the sequenced chicken ST6GalNAcI as comparedto the published sequence GenBank X74946. This difference results in aone amino acid difference of V251A (valine to alanine) in thepolypeptide. The DNA sequence also revealed one other silent mutationT233 (ACT to ACG) which was introduced by PCR primer. The rest of theDNA sequence was confirmed to be the same as the published sequence.

Example 5 Sialyltransferase Activity of N-Terminal Deletions of ChickenN-acetylgalactosamine-α2,6-sialytransferase (ST6GalNacI) in Sf9 CellsUsing Recombinant Baculovirus

This example describes the expression of four N-terminal deletions ofchicken N-acetylgalactosamine-α2,6-sialyltransferase (ST6GalNAcI), inSpodoptera frugiperda (Sf9) cells, using a pAcGP67 baculovirusexpression vector system. N-acetylgalactosamine-α2,6-sialyltransferase(ST6GalNAc I) transfers sialic acid from CMP-sialic acid, by an α2,6linkage, onto a N-acetylgalactosamine (GalNAc) residue, O-linked to athreonine or serine of a glycoprotein.

A viral stock expressing an N-terminal deletion of chicken ST6GalNAcIwas obtained. This viral stock was produced using a pVL1392 baculovirusexpression system (Blixt et al., 2002, J. Am. Chem. Soc.,124:5739-5746). The enzyme activity of multiple 10×1 L enzyme productionruns using this viral stock averaged 12 U/L.

Four N-terminal deletions of chicken ST6GalNAcI-Δ48, a truncation mutantthat begins at amino acid 49 of the full-length chicken ST6GalNAcI;A152, a truncation mutant that begins at residue 153 of the full-lengthchicken ST6GalNAcI; A225, a truncation mutant that begins at residue 226of the full-length chicken ST6GalNAcI; and A232, a truncation mutantthat begins at residue 233 of the full-length chicken ST6GalNAcI—werecreated using PCR. The resultant four PCR fragments contained ST6GalNAcIcoding sequences beginning with amino acids Q49, V153, L226 and T233,respectively. Sites of N-terminal deletions of the chicken ST6GalNAcIwere chosen based upon sequence similarities among the human, mouse andchicken ST6GalNAcI coding sequences (FIG. 28).

The Δ48 N-terminal deletion deletion mutant was designed to create acoding sequence initiating immediately after the predicted transmembranedomain. The transmembrane region of chicken ST6GalNAcI had previouslybeen predicted to be between amino acids 17 to 37 (Kurosawa et al.,1994, J. Biol. Chem., 269:1402-1409), but a hydropathy plot analysissuggested a transmembrane region between amino acids 26 and 48. The Δ152N-terminal deletion mutant was selected to create a truncation mutantthat included the portion of the stem region of chicken ST6GalNAcIenzyme that contained predicted areas of sequence similarity with thehuman and mouse enzymes (FIG. 31). The third N-terminal deletion mutant,Δ232, was created to resemble the ST6GalNAcI coding sequence aspublished by Blixt et al. (2002, J. Am. Chem. Soc., 124:5739-5746).

Initial activity assays indicated the Δ232 viral stock was inactive (seebelow). The ST6GalNAcI sequence contained in the original viral stockwas therefore analyzed. It was determined that additional N-terminalamino acids identical to those present in the wild type enzyme wereinadvertently donated to this truncation mutant sequence from themultiple cloning site of the vector, which included insertion of theamino acids DPK immediately N-terminal to Δ232. In the ST6GalNAcIfamily, the amino acids immediately upstream of Δ232 in all threesequences is (NED)FK (see Appendix 2). Therefore, a fourth N-terminaldeletion, Δ225, was created to be representative of the clone describedby Blixt et al. (2002, J. Am. Chem. Soc., 124:5739-5746).

A chicken ST6GalNAcI viral stock was produced using a vector, pVL1392,that contained a dog insulin secretion signal peptide. Other deletionsprepared for this study were cloned into a pAcGP67B vector (Pharmingen,San Diego, Calif.), which contains the glycoprotein 67 (gp67) secretionsignal peptide. The gp67 signal peptide was used as a stronger secretionsignal than the dog insulin secretion peptide. PCR reactions were set upas illustrated in Table 17.

TABLE 17 PCR Reactions for generation of truncation mutants. 5 μl 10xPCR Buffer 2 μl 10 mM dNTP 1 μl 5′ primer (10 pmol/μl) 1 μl 3′ primer(10 pmol/μl) 2 μl DMSO 1 μl DNA template (10 ng/μl) 0.5 μl   Herculase(Stratagene, Cat # 600260-51, Lot # 1220210) 37.5 μl   PCR grade H₂O ThePCR program was conducted under the following cycles: a) 95° C. 3minutes; b) 95° C., 45 sec; 42° C. 1 minute, 72° C. 1.5 minutes for 5cycles; c) 95° C., 45 sec; 57° C. 1 minute, 72° C. 1.5 minutes for 30cycles; d) 72° C. 10 minutes; e) 4° C. pause.

The PCR primer pair used to generate the Δ232 mutant was ch233BamHI2,5′-GATTCGGGATCCACGGAGCCACAGTGGGATTTTG-3′ (SEQ ID NO:60) and ch233XhoI,5′-GATCGCCTCGAGTCAGGATCTCTGGTAGAGCTTC-3 (SEQ ID NO:61). Isolated andconcentrated baculovirus DNA template was used for PCR. One microliterof template (600 ng/μl) was used for PCR. A 1002 bp PCR product wasproduced.

The PCR primer pair used to generate Δ48 was Δ48BamHI,5′-GGATCCCAAAGTATTGCACACATGCTACAAG-3′ (SEQ ID NO:62) and S566EcoRI,5′-GGCGAATTCTCACGATCTCTGGTAGAGTTTC-3′ (SEQ ID NO:63). The PCR primerpair used to generate the A 152 mutant was Δ152BamHI,5′-GGATCCGTTCCAGGTGTGGGAGAAGC-3′ (SEQ ID NO:64) and S566EcoRI (SEQ IDNO:63). The DNA template for both PCR fragments was plasmid DNApBluescript-chST6GalNAcI. For chST6GalNAc Δ48, a 1554 bp PCR product wasproduced. For chST6GalNAc 1-Δ152, a 1242 bp PCR product was produced.

The PCR primer pair used to generate the Δ225 mutant was A225BamHI,5′-GGATCCCTGAGGGCTGCTGACTTCAAGAC-3′ (SEQ ID NO:65) and5′-GGTGCTTAAGAGTAATGCTAGAGACCATCTCAAAGTAC-3′ (SEQ ID NO:66). The DNAtemplate was plasmid DNA pBluescript-chST6GalNAcI. The annealingtemperature for the first 5 cycles was 40° C. and for the last 30 cycleswas 53° C. For chST6GalNAc 1-Δ225, a 1023 bp PCR product was produced.

The PCR bands were electrophoresed and isolated by gel extraction. TheDNA was eluted in 20 μl 1×TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5). Toligate the isolated PCR products into a vector, ligation reactions wereconducted with each isolated DNA. For the chST6GalNAcI-Δ232 PCR product,the ligation reaction contained 4 μl PCR product, 1 μl salt solution,and 1 μl TOPO pCR4 (Invitrogen, Carlsbad, Calif.). The reaction wasincubated at room temperature for 15 minutes. For all other PCRproducts, the ligation reactions contained 4-7 μl PCR product, 1 μl pCR4Blunt vector (Invitrogen, Carlsbad, Calif.), 1 μl T4 DNA ligase Buffer,1 μl T4 DNA ligase, with the remaining volume up to 10 μl comprisingH₂O. The ligation reactions were incubated at 16° C. for 1 hour.

Subsequently, 6 μl of each ligation mixture was added to separate tubescontaining 50 μl of Top10 cells (Invitrogen, Carlsbad, Calif.).Incubations of each were performed on ice for 30 minutes, at 37° C. for1 minute, on ice for 2 minutes, adding 0-5 ml SOC then 37° C. 1 hour.After incubation, 200 μl of each incubation mixture was spread onkanamycin-containing plate. Approximately 100 colonies were generatedfor each transformation reaction.

Single colonies were selected and grown overnight at 37° C. in 3 mlmedium containing 50 μg/ml Kanamycin. The inserts were verified as beingcorrect by using pairwise restriction enzymes corresponding torestriction sites designed into the PCR primers.

The pAcGP67B vector and each insert in the pCRBlunt vector were digestedwith the restriction site-appropriate, pairwise restriction enzymes. Thedigested DNA was separated on 0.8% agarose gets. The corresponding bandswere excised with a surgical blade and DNA was extracted from the gelusing a MiniFlute Kit (Qiagen, Valencia, Calif.). The insert and vectorwere ligated together using T4 DNA ligase (in ratios ranging from 1:1 to6:1). The ligation mixtures were transformed into Top10 cells(Invitrogen, Carlsbad, Calif.) and spread on carbenicillin-containingplates. After overnight incubation at 37° C., several colonies werepicked and screened for the correct insert and vector for each plasmid.Subcloning procedures included pAcGP67B-Δ232 BamHI/EcoRI, pAcGP67B-Δ152and pAcGP67B-Δ48 BamHI/EcoRI and pAcGP67B-Δ225 BamHI/EcoRI.

In a 25 cm² Falcon flask (BD Bioscience, Franklin Lakes, N.J.), 1×10⁶Sf9 cells were seeded (50 to 70% confluence). Linearized BaculoGold DNA(BD Bioscience, Franklin Lakes, N.J.) (0.5 μg) was mixed with 2 pgrecombinant plasmid DNA and 100 μl of SF900 II SFM (Invitrogen,Carlsbad, Calif.) in a microfuge tube. In another tube, 6 μl ofcellfectin was mixed with 100 μl SF900 II SFM (Invitrogen, Carlsbad,Calif.). The two mixtures were combined and incubated at roomtemperature for 15 to 45 minutes. The medium in the flask was removedand Sf9 cells were covered with the DNA mixture. An additional 0.8 ml ofSF900 II SFM (Invitrogen, Carlsbad, Calif.) was added to the flask andincubated at 27° C. for 5 hours. After the incubation, the DNA mixtureand cellfectin were removed and 3 ml of fresh SF900 II SFM (Invitrogen,Carlsbad, Calif.) was added to the flask. The Sf9 cells in the flaskwere incubated, without shaking, for 5 days at 27° C. Visible infectionwas observed after 72 hours.

Following a 5 day incubation, the culture supernatant was cleared bycentrifugation at 1,000×g for 10 minutes. This supernatant was labeledthe Passage 1 (P1) viral amplification stock. One ml of the P1 viralstock was incubated with a 50 ml suspension culture of Sf9 cells (2×10⁶cells/ml). The incubation was conducted at 27° C., with stirring at 100rpm for 5 days. The culture was harvested by centrifugation in a Corningsterile conical centrifuge tube (Corning, Corning, N.Y.) at 5000 rpm(7,000×g) for 30 minutes at 4° C. and the resultant supernatant waslabeled the Passage 2 (P2) viral amplification stock.

Twelve ml of the P2 viral stock was incubated with a 150 ml suspensionculture of Sf9 cells (2×10⁶ cells/ml). The incubation was conducted at27° C., with stirring at 100 rpm for 5 days. The supernatant, isolatedas described for the P2 viral stock, was labeled the Passage 3 (P3)viral amplification stock. P1 and P2 were stored at −80° C. P3 wasstored at 4° C. in the dark. The titer of the recombinant baculoviruswas determined by plaque assay.

Recombinant protein was produced by infecting 200 ml of 2×10⁶ cells/mlSf9 cells with 25 ml of the P3 viral stock. The culture was incubated at27° C., with stirring at 100 rpm for 72 hours. The supernatant wasisolated as described for the P2 and P3 viral stocks.

The resultant supernatants were assayed for ability to catalyzesialylation of asialo bovine submaxillary mucin and to catalyze thetransfer of a sialic acid-polyethylene glycol conjugate to G-CSF(“sialylPEGylation” of G-CSF). More generically, the design and transferof a glycan-polyethylene glycol conjugate, or “glycoPEG” conjugate, toanother molecule is presented at length in International (PCT) PatentApplication No. WO03/031464 (PCT/US02/32263), which is incorporatedherein by reference in its entirety.

Radioactive assays were used to measure the transfer of ¹⁴C-sialic acidfrom ¹⁴C-CMP-sialic acid to asialo-bovine submaxillary mucin, asdescribed elsewhere herein.

TABLE 18 SialylPEGylation Assay G-CSF-O-GalNAc (0.4 mg/mL) 5.00 μl 125mM MnCl₂ 0.50 μl CMP-SA-PEG-20K 0.25 μl Chicken ST6GalNAc 1 5.00 μl 50mM MES pH 6.0 1.25 μl Total volume 12.0 μl

The sialylPEGylation reaction mixture was incubated at 33° C. withgentle shaking for 18 to 72 hours (as described below). Afterincubation, 2-5 III of 5×SDS Sample Buffer was added to each reactionmixture and the entire reaction mixture was subjected to electrophoresisin a 4-20% SDS-PAGE gradient gel. PEGylated G-CSF was detected by iodinestaining of the gel.

Using a DNA miniprep analysis, the pCRBlunt constructs were examined forinsert. The analysis demonstrated that pCRBlunt-chST6GalNAcI-Δ232BamHI/XhoI colonies # 2 to 15 # 6 were identified as containing thecorrect insert, pCRBlunt-chST6GalNAcI-Δ152 BamHI/EcoRI colonies # 1., #3 to # 6 were identified as containing the correct insert,pCRBlunt-chST6GalNAcI-Δ48 BamHI/EcoRI colonies # 1., # 2., # 3., # 5 and# 6 were identified as containing the correct insert, andpCRBlunt-chST6GalNAc 1-Δ225 EcoRI colonies A 2 were identified ascontaining the correct insert.

Using a DNA miniprep analysis, the subcloning constructs were examinedfor insert. The analysis demonstrated that pAcGP67B-chST6GalNAcI-Δ232BamHI/EcoRI colonies # 1 to # 4 were identified as containing thecorrect insert, pAcGP67B-chST6GalNAc 1-Δ152 BamHI/EcoRI colonies # 1 to# 4 were identified as containing the correct insert,pAcGP67B-chST6GalNAcI-Δ48 BamHI/EcoRI colonies # 1 to # 4 wereidentified as containing the correct insert, and pAcGP67B-chST6GalNAc1-Δ225 BamHI/EcoRI colonies # 1 to # 8 were identified as containing thecorrect insert.

The titers of recombinant baculovirus containing chicken ST6GalNAc lmutants were also determined. The Δ232 mutant had a titer of 8.50×10⁶,the Δ152 mutant had a titer of 2.28×10⁷, and the Δ48 mutant had a titerof 1.28×10⁷.

TABLE 19 Summary of Sialylation Activity in Radioactive Assay AverageAverage Activity (Units/L) Samples Activity (Units/L) (1:5 sample dil)Positive Control 40.6 51.5 K232 VS4-001 19.2** 19** Δ232 # 1 0 ND Δ232 #2 0 ND Δ48 # 1 35.6** 47** Δ48 # 2 34.5** 38.8** Δ152 # 1 39.5** 44.1**Δ152 # 2 39.9** 35.5** Δ225 27.877** ND (supernatant)

Purified enzyme, using K232 VS4-001, was used as a positive control. MOIused for protein production were as follows: Δ48 #1, 0.800; Δ152 #1,1.430; Δ232 #1, 0.531; Δ48 #2, 0.200; Δ152 #2, 0.356; Δ232 #2, 0.133.RSD less than 2.5%

Note: For the Δ232 viral stocks, since the activities were zero on Apr.8, 2004, they were not re-assayed on Apr. 26, 2004. Mutants with resultsmarked with a double asterisk (**) tested positive for sialylPEGylationactivity.

Example 6 Refolding of MBP-ST6GalNAcI Proteins

Eukaryotic ST6GalNAcI was fused to MPB. Briefly, five mouse ST6GalNAcIconstructs were generated: D32, E52, S127, S186, and S201. Eachconstruct was expressed behind the MBP-tag from the vector pcWin2-MBP,and differ in the extent of the ‘stem’ region included in the construct.D32 is the longest form, starting immediately downstream of thepredicted amino-terminal transmembrane domain. S201 is the shortest,beginning shortly before the predicted start of the conserved catalyticdomain.

In addition to the mouse constructs, human ST6GalNAcI K36 was alsoexpressed as a fusion with MBP. The human construct begins just afterthe transmembrane domain. DNA encoding human ST6GalNAcI from K36 to itsc-terminus was isolated by PCR using the existing baculovirus expressionvector as template, and cloned into the BamHI-XhoI sites within pcWin2MBP.

For reference, the sequences for MBP-mST6GalNAcI S127 andMBP-hST6GalNAcI K36 are included in FIG. 26. In addition, FIG. 38provides full length amino acid sequences for human ST6GalNAcI and forchicken ST6GalNAcI, and a sequence of the mouse ST6GalNAcI proteinbeginning at residue 32 of the native mouse protein.

Deletion mutants additional to those described above have been made anda complete list of preferred ST6GalNAcI for use in the invention isfound is Table 20. FIG. 35 provides a schematic of a number of preferredhuman ST6GalNAcI truncation mutants. FIG. 36 shows a schematic of MBPfusion proteins including the human ST6GalNAcI truncation mutants.

TABLE 20 ST6GalNAcI Mutants Truncation Site Mutation HUMAN Δ35 K36 Δ124K125 Δ257 S258 Δ35 K36 Δ72 T73 Δ109 E110 Δ133 M134 Δ170 T171 Δ232 A233Δ272 G273 CHICKEN Δ48 Q49 Δ152 V153 Δ225 L226 Δ226 R227 Δ232 T233 MOUSEΔ30 K31 Δ31 D32 Δ51 E52 Δ126 S127 Δ185 S186 Δ200 S201

FIG. 37 shows the position of paired and unpaired cysteine residues inthe human ST6GalNAcI protein. Single and double cysteine substitutionare also shown, e.g., C280S, C362S, C362T, (C280S+C362S), and(C280S+C362T).

Initial expression studies showed that the ST6GalNAcI fusion proteinswere expressed as insoluble proteins. To recover active recombinantenzyme, the inactive, insoluble proteins were isolated and refolded asdescribed:

Logarithmically growing 0.5 L cultures of JM109 cells bearing eitherpcWin2-MBP-mST6GalNAcI D32, E52, S127, S186, or pcWin2-MBP-hST6GalNAcIK36 were induced with 1 mM IPTG overnight at 37° C. Cells were collectedby centrifugation, and lysed by mechanical disruption in amicrofluidizer in 100 mL of 20 mM Tris pH8, 5 mM EDTA. Insoluble matterwas collected by centrifugation at 7000×g for 20 minutes. Thesupernatants were discarded, and the pellets were washed with a highsalt buffer (20 mM Tris pH 7.4, 1M NaCl, 5 mM EDTA), detergent buffer(25 mM Tris pH 8, 1% Na-deoxycholate, 1% Triton×100, 100 mM NaCl, 5 mMEDTA), and TE (10 n Tris pH 8, 1 mM EDTA). Each wash was in 100 mL, andthe pellet was collected by centrifugation as described above. Followingthe washing, the inclusion body pellets were aliquoted and stored at−80° C.

To screen for conditions that allow proper refolding and thus recoveryof ST6GalNAcI activity, aliquots of the mouse and human ST6GalNAcIfusion protein inclusion bodies were solubilized in 6M guanidine, 10 mMDTT, 1×TBS. Protein concentration was normalized by Bradford assay, andthe solubilized proteins were transferred to a series of commerciallyavailable protein refolding buffers. Refolds were carried out in 0.25 mLat 0.2 mg/mL overnight at 4° C. in a 96-well plate with shaking. Therefolds were transferred to a 96-well dialysis plate (25000 MWCO) anddialyzed against 1×TBS, 0.05% Tween-80 for four hours at 4° C., followedby overnight dialysis against 10 mM BisTris pH 7.1, 100 mM NaCl, 0.05%Tween-80 at 4° C.

Refolded recombinant ST6GalNAcI fusion proteins were tested for activityin a 384-well solid phase activity assay. Briefly, the activity assaydetects the ST6GalNAcI-mediated transfer of a biotinylated sialic acidfrom biotinylated CMP-NAN to the surface of an asialo-bovinesubmaxillary mucin-coated well in a 384-well plate. Each reaction (13.5μL refold+1.5 μL 10× reaction buffer) was performed in quadruplicate.10× reaction buffer was 0.2M BisTris ph 6.7, 25 mM MgCl2, 25 mM MnCl2,0.5% Tween-80, and 1 mM donor. After overnight incubation at 37° C., theplate was washed with excess 1×TBS, 0.05% Tween-20, and biotin detectedwith europium-labeled streptavidin as per manufacturer's instructions(Perkin Elmer). Europium fluorescence levels retained on the plate,indicative of ST6GalNAcI activity, were documented with a Perkin ElmerVictor3V plate reader, and expression and activity results aresummarized in Table 21. Three of the refolded ST6GalNAcI fusion proteinshad detectable activity.

TABLE 21 Summary of refolded ST6GalNAcI fusion proteins tested foractivity by solid phase assay. Refolded protein Refolded proteindetected by SDS- activity detected Construct PAGE by solid phase assayMBP-mST6GalNAcI D32 + − MBP-mST6GalNAcI E52 ++ − MBP-mST6GalNAcI S127+++ + MBP-mST6GalNAcI S186 +/− +/− MBP-hST6GalNAcI K36 +/− +

In summary, four N-terminal deletions of chicken ST6GalNAcI weresuccessfully expressed in Sf9 cells as secreted, active enzymes. Maximalactivity levels for the four active clones varied, with K232 VS4-001 at19 U/L, Δ48 at 47 U/L, Δ152 at 44.1 U/L, and Δ225 at 27.9 U/L.Additionally, mutant chicken ST6GalNAcI produced in Δ48, Δ152 and Δ225viral stocks were equally able to sialylPEGylate GalNAc−O-G-CSF (FIGS.32 and 33).

Example 7 Generation of Additional Human ST6GalNAcI Proteins

Cloning hST6GalNAcI truncations: The following oligos:hST6-T73-hST6-G273 and hST6CooH were used to amplify various humanST6GalNAcI truncations

TABLE I Truncation oligos for hST6GalNAcI. hST6-T735′TATTGGATCCACAACCATCTATGCAGAGCCAG hST6-E1105′TATTGGATCCGAGGAGCAGGACAAGGTGCCC hST6-M1345′TATTGGATCCATGGTGAACACACTGTCACCCA hST6-T1715′TATTGGATCCACCAGGAAGCTGACGGCCTCCA hST6-A2335′TATTGGATCCGCCACCCCACCCCCTGCCCCTT hST6-G2735′TATTGGATCCGGAGGCCTTCAGACGACTTGCC hST6-CooH5′GCGCTCTAGATCAGTTCTTGGCTTTGGCAGTTCC The BamHI restriction site foroligos, hST6-T73 - G273 and XbaI restriction site for hST6-CooH oligowere underlined.

Template DNA: phST6GalNAcI K36 (the plasmid carrying Δ35 truncation ofhST6GalNAcI gene)

PCR reactions: Fifty μl reactions were carried out using Herculase®Enhanced DNA polymerase (Stratagene) under PCR conditions: 30 cycles:92° C., 45 s; 61° C., 1 min; 72° C., 3 min; and 4 cycles: 92° C., 45 s;61° C., 1 min; 72° C., 10 min.

Agarose gel analysis: Three μl aliquots from the PCR reactions wereanalyzed in 1% agarose gel in TAE buffer stained with EtBr.

Cloning hST6GalNAcI truncations: The PCR amplified DNA fragments werepurified using Millipore Ultrafree DA cartridges from the agarose geland concentrated using Amicon microcon YM-100 filters. One to two ulaliquots from purified DNA fragments were used in Zero Blunt® TOPO® PCRcloning kit (Invitrogen). The reactions were transformed into competentTOP10 E. coli cells (Invitrogen). The following colonies obtained after50 μl transformants were introduced onto Martone Agar Kan50 plates(Teknova)

Truncation # of colonies K36 6 T73 25 E110 9 M134 15 T171 34 A233 32G273 4

The plasmids DNAs were obtained from the cultures after growing theselected colonies (4-5 from each truncation) in 5 mls of Martone L-Brothliquid media (Teknova) supplemented with 50 μg/ml Kanamycin.

Screening hST6GalNAcI clones: The plasmid DNAs were purified from 4 mlovernight cultures using Wizards Plus SV Minipreps DNA purificationsystem. The purified plasmids (10 μl) were digested with BamHI and XbaIrestriction enzymes followed by agarose gel analysis [1.2% E-gel(Invitrogen)] to confirm the correct inserts (truncations).

The hST6GalNAcI truncations above were cloned into baculovirusexpression vector, pAcGP67B, and expressed in SF9 insect cell culture.ST6GalNAcI activities were determined in the samples obtained from theinfected cultures and results are shown in FIG. 38. Each of thetruncated human ST6GalNAcI proteins had detectable activity afterexpression in the bacculoviral system. The hST6-E 10 protein had thehighest activity.

The hST6GalNAcI truncations are shown in FIG. 39. The figure also showsan alignment of the human sequence with the mouse and chicken proteinsand identifies identical and conserved amino acid residues between theproteins.

Example 8 Truncated ST6GalNAcI Proteins that Comprise SBD Sequences

N-acetylgalactosamine-α-2,6-sialyltransferase (ST6GalNAc 1) transferssialic acid from CMP-sialic acid, by an α-2,6 linkage, onto aN-acetylgalactosamine (GalNAc) residue, O-linked to a threonine orserine of a glycoprotein.

This report describes the cloning and expression of the SBD tag at theN-terminal and the C-terminal of the human (SBD-K36, K36-SBD) and mouse(SBD-S127, S127-SBD) ST6GalNAcI in Spodoptera frugiperda (Sf9) cells,using the pAcGP67 baculovirus expression system.

All four viral stocks were used to infect SF9 cells (150 mL scale) for96 hours and the resultant supernatants were isolated on β-cyclodextrincolumn, concentrated and assayed for both sialylation of asialo bovinesubmaxillary mucin and sialylPEGylation of G-CSF.

METHODS 1. Construct Design

A three way fusion among the gp67 secretion peptide, the ST6GalNAcIcoding sequence and the SBD coding sequence was constructed. Based onthe restriction maps (Appendix 4) of ST6GalNAcI and S B D and themultiple cloning sites in pAcGP67B vector, NcoI/NotI/BgtII was chosenfor cloning the SBD-ST6GalNAcI constructs and BamHI/NotI/BglII waschosen for cloning ST6GalNAcI-SBD constructs. The NotI site introducedfour amino acids (WRPP or RRPP) between the SBD and ST6GalNAcI codingsequences. This extension could help to separate these two proteindomains. The SBD gene codon optimized for E. coli was not used in thiswork. The original A. niger SBD coding sequence was chosen, as it wasdetermined that the codon codon bias of SF9 cells would be closer tothat of the eukaryotic A. niger as opposed to the prokaryotic E. coli.

2. PCR Reactions 5 μl 10x PCR Buffer 2 μl 10 mM dNTP 1 μl 5′ primer (10pmol/μl) 1 μl 3′ primer (10 pmol/μl) 2 μl DMSO 1 μl DNA template (10ng/μl)

0.5 ul Herculase (Stratagene, Cat # 600260-51)

37.5 μl PCR grade H₂O

The PCR Program used for K36 and S127 was a) 95° C. 3 min; b) 95° C., 45sec; 42° C. 45 sec, 72° C. 1.5 min for 5 cycles; c) 95° C., 45 sec; 54°C. 45 sec, 72° C. 1.5 min for 30 cycles; d) 72° C. 10 min; e) 4° C.pause. (LL774, pg 51). PCR were performed using a T3 Thermocycler.

The PCP Program used for SBD was a) 95° C. 3 min; b) 95° C., 45 sec; 40°C. 45 sec, 72° C. 1 min for 5 cycles; c) 95° C., 45 sec; 55° C. 45 sec,72° C. 1 min for 30 cycles; d) 72° C. 10 min; e) 4° C. pause. (LL774, pg51). PCR were performed using a T3 Thermocycler.

3. Gel Extraction

A MinElute Gel Extraction Kit was used to isolate all the PCR bands. TheDNA was eluted in 20 μl 1×TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5).

4. pCRBlunt Ligation and Transformation4.5 μl PCR product

1.0 μl Salt Solution

0.5 μl TOPO pCR4

The reaction was incubated at room temperature for 9 min.

Two microliters of each ligation mixture was added to separate eppendorftubes containing 25 μl of Top 10 cells. The following incubations ofeach were performed: on ice 5 min, 42° C. 45 sec, on ice 2 min, adding0.1 mL SOC then 37° C. 1 hour. After incubation, 120 μl of the mixturewas spread on Kanamycin plate. About 7 to 70 colonies were generated foreach transformation (LL774, pg 51).

Plasmid DNA Minipreps

Single colonies were picked and grown, overnight at 37° C. in 2 mLterrific broth medium containing 50 μg/mL Kanamycin. The correct insertwas checked with the pairwise restriction enzymes whose sites weredesigned into the PCR primers.

5. Subcloning

The pAcGP67B vector and each insert in the pCRBlunt vector were digestedwith the appropriate, pairwise restriction enzymes. The digested DNA wasseparated on 0.8% agarose gels. The corresponding bands were cut outwith a surgical blade and DNA was extracted from the gel using theMiniElute Kit. The insert and vector were ligated together using T4 DNAligase. The ligation mixture was transformed into Top10 cells and spreadon ampicillin (carbenicillin) plates. After overnight incubation at 37°C., several colonies were picked and screened for the correct insert andvector for each plasmid

6. Cotransfection

In a 25 cm² falcon flask, 1×10⁶ Sf9 cells were seeded (50 to 70%confluence). Linearized BaculoGold DNA (0.5 μg) was mixed with 2 μgrecombinant plasmid DNA and 100 μL of SF900 II SFM in an eppendorf. Inanother tube, 6 μL of cellfectin was mixed with 100 μL SF900 II SFM. Thetwo mixtures were combined and incubated at room temperature for 15 to45 min. The medium in the flask was removed and Sf9 cells were coveredwith the DNA mixture. An additional 0.8 mL of Sf900 μl SFM was added tothe flask and incubated at 27° C. for 4 hours. After the incubation, theDNA mixture and cellfectin were removed and 3 mL of fresh Sf900 If SFMwas added to the flask. The S19 cells in the flask were incubated,without shaking, for 3 days at 27° C. Visible infection could be seenafter 72 hours (LL774, pg 89).

7. Recombinant Baculovirus amplification

Following the 3 day incubation, the culture supernatant was cleared bycentrifugation at 1,000×g for 10 min. This supernatant was labeled thePassage Zero (P0) viral amplification stock.

P0 viral stock (0.5 mL) was incubated with a 50 mL suspension culture ofSf9 cells (1×10⁶ cells/mL). The incubation was done at 27° C., withstirring (100 rpm) for 3 days. The culture was harvested bycentrifugation in a Corning sterile conical centrifuge tube at 5000 rpm(7,000×g) for 30 min at 4° C. and the resultant supernatant was labeledthe Passage 1 (P1) viral amplification stock (LL774, pg 96).

Three mL of the P2 viral stock was incubated with a 150 mL suspensionculture of Sf9 cells (1×10⁶ cells/mL). The incubation was done at 27°C., with stirring (100 rpm) for 66 hours. The supernatant, isolated asdescribed for the P1 viral stock, was labeled the Passage 2 (P2) viralamplification stock (LL774, pg 96, 103).

P0 stored at −80° C. P1 and P2 were stored at 4° C. in the dark. Thetiter of the recombinant baculovirus at P2 was determined by plaqueassay.

8. Low MOI Protocol LL774, pg 120 Materials:

30 mL of Sf9 cells in 250 mL shake flask. Total flasks: 10.The targeting cell concentration is: 1.5E6 cells/mL.The targeting MOI is: 5E-4 to 5E-8.Baculovirus virus:

SBD-K36 2.55 × 10⁷ pfu/mL K36-SBD 2.25 × 10⁷ pfu/mL

Calculation:

Total cells: 1.5e6×30-45e6Total virus for the highest MOI:SBD-K36 (5E-4×45e6)/2.55E7=22500/2.55E7=0.88 μlK36/SBD (5E-4×45e6)/2-25E7=22500/2.25E7=1.00 μl

Dilution Procedures:

Dilute virus by:

-   -   8.8 μl SBD-K36 virus+1 mL Sf90011 SFM for MOI 5e-3        10.0 μl K36-SBD virus+1 mL Sf 900II SFM for MOI 5e-3

0.2 mL 5E-3+1.8 mL Sf 900II SFM for MOI 5E-4 0.2 mL 5E-4+1.8 mL Sf 900IISFM for MOI 5E-5 0.2 mL 5E-5+1.8 mL Sf 900II SFM for MOI 5E-6 0.2 mL5E-6+1.8 mL Sf 900 II SFM for MOI 5E-7 0.2 mL 5E-7+1.8 mL Sf 900 II SFMfor MOI 5E-8 Experiments:

Start experiments by adding 1 mL of each dilution to 30 mL of Sf 9cells.Check cell concentration and take 1 mL sample for radioactive assay onDay 4, Day 5, Day 6 and Day 7.Summary results.

Note:

-   -   The actual starting cell concentration is 1.47E6.    -   The cells were in PSG16.

9. Protein Production

Recombinant protein was produced by infecting 150 mL of 1.5×10⁶ cells/mLSf9 cells with 75 μl of the P2 viral stock. The culture was incubated at27° C., with stirring (100 rpm) for 96 hours. The supernatant wasisolated as described for the P1 and P2 viral stocks. The MOI used forinfection were: SBD-K36, 0.0085; K36-SBD, 0.0075; SBD-S127, 0.013;S127-SBD, 0.013 (LL774 pg 103, 120).

10. Purification of ST6GalNAcI Enzyme Using SBD Tag on β-CyclodextrinColumn

Human and mouse ST6GalNAcI fused with SBD tag was isolated from Sf9 cellsupernatant by passage through a β-cyclodextrin column. Either 22.5 mUor 182.4 mU of SBD-human or mouse ST6GalNAcI, respectively, were loadedonto separate β-cyclodextrin columns (bed volume 7.5 mL) at about 0.4mL/min at 4° C. The column was washed with 80 to 100 mL of Wash Buffer(1×PBS pH 7.4 Fisher Cat # BP-399-500). The bound enzyme was eluted fromthe column by Elution Buffer (3 mM β-cyclodextrin Sigma Cat # C4767 inWash Buffer). Twelve fractions of 1 to 2 mL were collected. The elutionprofile was recorded as OD₂₈₀. The peak fractions were pooled andconcentrated using a VIVASPIN 6 mL or 20 mL concentrator on Joanncentrifuge at 4° C. for about 30 min at 7500 rpm (−6000 g). One mL ofWash Buffer was added to the concentrator at this point and continued toconcentrate to a final volume of 100 to 200 μL. The concentrated productwas tested for sialyation and sialylPEGylation.

The β-cycledextrin column was regenerated using 1 M NaCl in Wash bufferand then equilibrated with Wash Buffer or soaked in 0.5 M NaOHovernight. The column was next washed with H₂O until the pH reached 7.0and then equilibrated with Wash Buffer.

11. Sialylation Radioactive Assay

Radioactive assays measured the transfer of ¹⁴C_Sialic acids from¹⁴C-CMP-sialic acid to asialo-bovine submaxillary mucin (see DR-518-04for details).

12. SialylPEGylation Assay LL774, pg 163 G-CSF-O-GalNAc (0.4 mg/mL) 10.0μL 125 mM MnCl₂  0.5 μL CMP-SA-PEG-20K  0.5 μL ST6GalNAcl  2.0 μL 100 mMBis-Tris pH 6.5 10.0 μL Total volume 23.0 μL

The reaction mixture was incubated, at 33° C., with no shaking for 66hours.

After incubation, 2.5 μL of 5×SDS Sample Buffer (no DTT) was added to 5μl of reaction with 5 μl of water and was loaded onto a 4 to 20%SDS-PAGE gradient gel without heating the samples

PEGylated G-CSF was detected by iodine staining of the gel.

To the rest reaction mixture, 42 μl of water was added and the samplewas analyzed by HPLC.

GalNActylation reaction: G-CSF (~1 mg/mL in 40 mM Bis-Tris pH 6.5) 140μl  100 mM MnCl₂ 3 μl  30 mM UDP-GalNAc 9 μl 100 mM Bis-Tris pH 6.5 113μl  GalNAcT2 5 μl

33° C. no shaking for 2 days.

Results 1. PCR Results

The correct length PCR bands of K36 BamHI/NotI, S127BamHI/NotI, K36NotI/BglII, S127 NotI/BglLL and SBD NotI/BglII were generated.

2. Cloning Results

The correct length clones were generated.

3. Titers of Recombinant (P2) Baculovirus Stocks of the ST6GalNAcIClones

SBD-K36 2.55 × 10⁷ K36-SBD 2.25 × 10⁷ SBD-S127 3.85 × 10⁷ S127-SBD 3.95× 10⁷

4. Summary of Sialylation Activity in Radioactive Assay

Average Average Activity (Units/L) Activity (Units/L) Samples Assay dateSep. 2, 2004 Assay date Sep. 27, 2004 SBD-K36 P2 0.512 — K36-SBD P20.279 — SBD-S127 P2 0.206 — S127-SBD P2 0.201 — SBD-K36 175 mL — 0.851K36-SBD 175 mL — 0.619 SBD-S127 175 mL — 0.264 S127-SBD 175 mL — 0.397SBD-K36 300 mL — 0.638 K36-SBD 300 mL — 0.414

5. Ultra Low MOI Study Results

ST6GalNAc 1 Low MOI Study Activity in Unit/L Day MOI 4 5 6 7 5.00E−04SBD-K36 1.222 1.311 1.304 1.532 5.00E−05 SBD-K36 1.934 1.786 1.779 1.7515.00E−06 SBD-K36 2.815 2.632 2.407 1.873 5.00E−07 SBD-K36 0.900 1.8313.087 3.253 5.00E−08 SBD-K36 0.080 0.247 0.920 1.256 5.00E−04 K36-SBD0.638 0.559 0.796 0.630 5.00E−05 K36-SBD 0.923 0.673 1.387 0.6965.00E−06 K36-SBD 0.695 0.945 1.012 0.956 5.00E−07 K36-SBD 0.901 1.4562.264 1.798 5.00E−08 K36-SBD 0.136 0.175 0.494 0.439 Sf 9 (E6 cells/mL)Day MOI 4 5 6 7 5.00E−04 SBD-K36 3.65 1.85 2.30 1.00 5.00E−05 SBD-K366.25 3.60 3.10 1.75 5.00E−06 SBD-K36 9.10 5.10 2.90 1.85 5.00E−07SBD-K36 11.40 3.05 8.80 6.85 5.00E−08 SBD-K36 11.60 12.00 2.60 7.705.00E−04 K36-SBD 3.75 1.40 1.70 0.45 5.00E−05 K36-SBD 4.60 2.40 2.501.15 5.00E−06 K36-SBD 8.90 4.50 4.10 2.15 5.00E−07 K36-SBD 9.90 8.5011.10 6.75 5.00E−08 K36-SBD 11.90 11.00 2.30 5.85

Starting cell concentration was 1.47E6

cells/mL, LL-774 pg 120

6. Summary of the Purification of Human and Mouse ST6GalNAc 1 SBD FusionProteins on β-Cyclodextrin Column.

Species SBD-K36 K36-SBD SBD-S127 S127-SBD Date Sep. 14, Sep. 17, 2004Sep. 15, 2004 Sep. 16, 2004 2004 Load (mu/mL) 1.459 0.826 0.167 0.297Load (mL) 125 125 135 138 Load (mu) 182.375 103.25 22.545 40.986 FT(mu/mL) 0.452 0.015 0.016 0.005 FT (mL) 125 125 135 138 FT (mu) 52.51.875 2.16 0.69 Wash (mu/mL) 0.091 0.029 0.033 −0.028 Wash (mL) 80 80100 105 Wash (mu) 7.28 2.32 3.3 0 Bound (%) 67.22%  95.94%  75.78% 98.32%  Bound (mu) 122.58 99.055 17.085 40.296 Elute (mu/mL) 122.90822.928 5.510 15.943 Elute (mL) 0.26 0.42 0.46 0.35 Elute (mu) 31.9569.630 2.535 5.58 Recovered 26.1%  9.7% 14.8% 13.8% PEG-gCSF 72.9% 30.7%11.0%  9.3%

Two human ST6GalNAcI fusion constructs (SBD-K36 and K36-SBD) and twomouse ST6GalNAcI fusion constructs (SBD-127 and 127-SBD) have beensuccessfully expressed in Sf9 cells as secreted, active enzymes andpurified on a β-cyclodextrin column using their SBD tags.

The activity levels of purified, concentrated samples of the four activeclones were:

SBD-K36 122.9 U/L  K36-SBD 22.9 U/L SBD-S127  5.5 U/L S127-SBD 15.9 U/L

All four enzymes were able to sialylPEGylate G-CSF.

Using ultra low MOIs, the activity expression of human ST6GalNAcI-SBDproteins was increased, with an MOI of 5e-7 with SBD-6 on Day 6 and Day7 giving activity of 3-09 U/L and 3.25 U/L respectively and with an MOIof 5e-7 with K36-SBD on Day 6 giving activity was 2.26 U/L.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. An isolated truncated ST6GalNAcI polypeptide, wherein said truncatedST6GalNAcI polypeptide is lacking all or a portion of the ST6GalNAcIsignal domain, and wherein said ST6GalNAcI polypeptide is selected fromthe group consisting of a human ST6GalNAcI polypeptide and a chickenST6GalNAcI polypeptide, with the proviso that said polypeptide is not achicken ST6GalNAcI polypeptide truncation mutant lacking amino acidresidues 1-232.
 2. The isolated truncated ST6GalNAcI polypeptide ofclaim 1, wherein said truncated ST6GalNAcI polypeptide is furtherlacking all or a portion of the ST6GalNAcI transmembrane domain.
 3. Theisolated truncated ST6GalNAcI polypeptide of claim 2, wherein saidtruncated ST6GalNAcI polypeptide is further lacking all or a portion ofthe ST6GalNAcI stem domain.
 4. The isolated truncated ST6GalNAcIpolypeptide of claim 1, wherein said truncated ST6GalNAcI polypeptidehas at least 90% identity with a polypeptide selected from the groupconsisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, Δ35 of the humansequence shown in FIG. 31, Δ72 of the human sequence shown in FIG. 31,Δ109 of the human sequence shown in FIG. 31, Δ133 of the human sequenceshown in FIG. 31, Δ170 of the human sequence shown in FIG. 31, Δ232 ofthe human sequence shown in FIG. 31, Δ272 of the human sequence shown inFIG. 31, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and Δ225 of thechicken sequence shown in FIG.
 31. 5. The isolated truncated ST6GalNAcIpolypeptide of claim 1, wherein said truncated ST6GalNAcI polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, Δ35 of the human sequenceshown in FIG. 31, Δ72 of the human sequence shown in FIG. 31, Δ109 ofthe human sequence shown in FIG. 31, Δ133 of the human sequence shown inFIG. 31, Δ170 of the human sequence shown in FIG. 31, Δ232 of the humansequence shown in FIG. 31, Δ272 of the human sequence shown in FIG. 31,SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and Δ225 of the chickensequence shown in FIG.
 31. 6. The isolated truncated ST6GalNAcIpolypeptide of claim 1, wherein said truncated ST6GalNAcI polypeptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, Δ35 of the human sequenceshown in FIG. 31, Δ72 of the human sequence shown in FIG. 31, Δ109 ofthe human sequence shown in FIG. 31, Δ133 of the human sequence shown inFIG. 31, Δ170 of the human sequence shown in FIG. 31, Δ232 of the humansequence shown in FIG. 31, Δ272 of the human sequence shown in FIG. 31,SEQ ED NO:28, SEQ ID NO:30, SEQ ID NO:32, and Δ225 of the chickensequence shown in FIG.
 31. 7. An isolated chimeric polypeptidecomprising a tag polypeptide covalently linked to the isolated truncatedST6GalNAcI polypeptide of claim
 1. 8. The isolated chimeric polypeptideof claim 7, wherein said tag polypeptide is selected from the groupconsisting of a maltose binding protein, a histidine tag, a Factor IXtag, a glutathione-S-transferase tag, a FLAG-tag, and a starch bindingdomain tag.
 9. An isolated nucleic acid that comprises and nucleic acidsequence that encodes isolated truncated ST6GalNAcI polypeptide of claim1 or claim
 4. 10. The isolated nucleic acid of claim 9, said nucleicacid further comprising a promoter/regulatory sequence operably linkedthereto.
 11. An expression vector comprising the isolated nucleic acidof claim
 9. 12. A recombinant host cell comprising the isolated nucleicacid of claim
 11. 13. A recombinant cell of claim 12, wherein saidrecombinant cell is a eukaryotic cell or a prokaryotic cell.
 14. Therecombinant cell of claim 13, wherein said eukaryotic cell is selectedfrom the group consisting of a mammalian cell, an insect cell and afungal cell.
 15. The recombinant cell of claim 14, wherein said insectcell is selected from the group consisting of an SF9 cell, an SF9+ cell,an Sf21 cell, a HIGH FIVE cell or Drosophila Schneider S2 cell.
 16. Therecombinant cell of claim 13, wherein said prokaryotic cell is selectedfrom the group consisting of an E. coli cell and a B. subtilis cell. 17.A method of producing a truncated ST6GalNAcI polypeptide, the methodcomprising growing the recombinant cell of claim 13 under conditionssuitable for expression of the truncated ST6GalNAcI polypeptide.
 18. Amethod of catalyzing the transfer of a sialic acid moiety to an acceptormoiety comprising incubating the polypeptide of claim 1 with a sialicacid moiety and an acceptor moiety, wherein said polypeptide mediatesthe covalent linkage of said sialic acid moiety to said acceptor moiety,thereby catalyzing the transfer of a sialic acid moiety to an acceptormoiety.
 19. A method of catalyzing the transfer of a sialic acid moietyto an acceptor moiety comprising incubating the polypeptide of claim 1with a cytidinemonophosphate-sialic acid (CMP-NAN) sialic acid donor andan asialo bovine submaxillary mucin acceptor moiety, wherein saidpolypeptide mediates the transfer of said sialic acid moiety from saidCMP-NAN sialic acid donor to said asialo bovine submaxillary mucinacceptor, thereby catalyzing the transfer of a sialic acid moiety to anacceptor moiety.
 20. A method of catalyzing the transfer of a sialicacid moiety to an acceptor moiety comprising incubating the polypeptideof claim 1 with a cytidinemonophosphate-sialic acid (CMP-NAN) sialicacid donor and a polypeptide acceptor, wherein said polypeptide acceptoris selected from the group consisting of erythropoietin, human growthhormone, granulocyte colony stimulating factor, interferons alpha,-beta, and -gamma, Factor IX, follicle stimulating hormone,interleukin-2, erythropoietin, anti-TNF-alpha, and a lysosomalhydrolase.
 21. The method of claim 20, wherein said polypeptide acceptoris a glycopeptide.
 22. The method of claim 19 or claim 20, furtherwherein said sialic acid moiety comprises a polyethylene glycol moiety.23. The method of claim 19 or claim 20, wherein said method is carriedout on a commercial scale.